Modifying the specificity of non-coding rna molecules for silencing gene expression in eukaryotic cells

ABSTRACT

A method of modifying a gene encoding or processed into a non-coding RNA molecule having no RNA silencing activity in a eukaryotic cell, with the proviso that said eukaryotic cell is not a plant cell, is disclosed. The method comprising introducing into the eukaryotic cell a DNA editing agent conferring a silencing specificity of said non-coding RNA molecule towards a target RNA of interest. A method of modifying a gene encoding or processed into a RNA silencing molecule to a target RNA in a eukaryotic cell is also disclosed. Methods of disease prevention and treatment, methods of inducing cell apoptosis and methods of generating a eukaryotic non-human organism are also disclosed.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to modifyinggenes that encode or are processed into non-coding RNA molecules,including RNA silencing molecules and, more particularly, but notexclusively, to the use of same for silencing endogenous or exogenoustarget RNA of interest in eukaryotic cells which are not plant cells.

Among the approximately 25,000 annotated genes in the human genome,mutations in over 3,000 have already been linked to disease phenotypesand more disease relevant genetic variations are being uncovered at astaggeringly rapid pace. Emerging therapeutic strategies that can modifynucleic acids within disease-affected cells and tissues have potentialfor the treatment of monogenic, highly penetrant diseases, such asSevere Combined Immunodeficiency (SCID), hemophilia and certain enzymedeficiencies, owing to their well-defined genetics and often lack ofsafe, effective alternative treatments. Two of the most powerful genetictherapeutic technologies developed thus far are gene therapy, whichenables restoration of missing gene function by viral transgeneexpression, and RNA interference (RNAi), which mediates repression ofdefective genes by knockdown of the target mRNA.

Gene therapy has been used to successfully treat monogenic recessivedisorders affecting the hematopoietic system, such as SCID andWiskott-Aldrich syndrome, by semi-randomly integrating functional genesinto the genome of hematopoietic stem/progenitor cells [Gaspar et al.,Sci. Transl. Med. (2011) 3: 97ra79; Howe et al., J. Clin. Invest. (2008)118: 3143-3150]. RNAi has been used to repress the function of genesimplicated in cancer, age-related macular degeneration and transthyretin(TTR)-related amyloidosis, among others in clinical trials. Despitepromise and recent success, gene therapy and RNAi have limitations thatpreclude their utility for a large number of diseases. For example,viral gene therapy may cause mutagenesis at the integration site andresult in dysregulated transgene expression [Howe et al. (2008), supra].Meanwhile, the use of RNAi is limited to targets for which geneknockdown is beneficial. Also, RNAi often cannot fully repress geneexpression due to the transient nature of the delivered siRNA and thelack of silencing amplification mechanisms like in plants or nematodes,and is therefore, unlikely to provide a benefit for diseases in whichcomplete repression of gene function is necessary for therapy. Thecurrent main obstacle of RNA-based therapeutics is efficient andeffective RNA delivery into cells. Although some delivery agents canenhance therapeutic RNA endocytosis, only a very small fraction, lessthan 0.01%, escapes from the endosomes and are biologically active[Steven F Dowdy, Nature Biotechnol (2017) 35, 222-229].

Recent advances in genome editing techniques have made it possible toalter DNA sequences in living cells by editing only a few of thebillions of nucleotides in the cells of human patients. In the pastdecade, the tools and expertise for using genome editing in humansomatic cells and pluripotent cells have increased to such an extentthat the approach is now being developed widely as a strategy to treathuman disease. The fundamental process depends on creating asite-specific DNA double-strand break (DSB) in the genome and thenallowing the cell's endogenous DSB repair machinery to fix the break(such as by non-homologous end-joining (NHEJ) or homologousrecombination (HR)) in which the latter can allow precise nucleotidechanges to be made to the DNA sequence [Porteus, Annu Rev PharmacolToxicol. (2016) 56:163-90].

Three primary approaches use mutagenic genome editing (NHEJ) of cells aspotential therapeutics: (a) knocking out functional genetic elements bycreating spatially precise insertions or deletions, (b) creatinginsertions or deletions that compensate for underlying frameshiftmutations; hence reactivating partly- or non-functional genes, and (c)creating defined genetic deletions. Although several differenttherapeutic applications use editing by NHEJ, the broadest applicationsof therapeutic editing will probably harness genome editing byhomologous recombination (HR), although a rare event is highly accurateas it relies on a template to copy the correct sequence during therepair process.

Currently the four major types of therapeutic applications toHR-mediated genome editing are: (a) gene correction (i.e. correction ofdiseases that are caused by point mutations in single genes), (b)functional gene correction (i.e. correction of diseases that are causedby mutations scattered throughout the gene), (c) safe harbor geneaddition (i.e. when precise regulation is not required or when supraphysiologic levels of a therapeutic transgene are desired), and (d)targeted transgene addition (i.e. when precise regulation is required)[Porteus (2016), supra]. Previous work on genome editing of RNAmolecules in various eukaryotic organisms (e.g. murine, human, shrimp,plants), focused on knocking-out miRNA gene activity or changing theirbinding site in target RNAs, for example:

With regard to genome editing in human cells, Jiang et al. [Jiang etal., RNA Biology (2014) 11 (10): 1243-9] used CRISPR/Cas9 to depletehuman miR-93 from a cluster by targeting its 5′ region in HeLa cells.Various small indels were induced in the targeted region containing theDrosha processing site (i.e. the position at which Drosha, adouble-stranded RNA-specific RNase III enzyme, binds, cleaves andthereby processes primary miRNAs (pri-miRNAs) into pre-miRNA in thenucleus of a host cell) and seed sequences (i.e. the conservedheptametrical sequences which are essential for the binding of the miRNAto mRNA, typically situated at positions 2-7 from the miRNA 5′-end).According to Jiang et al. even a single nucleotide deletion led tocomplete knockout of the target miRNA with high specificity.

With regard to genome editing in murine species, Zhao et al. [Zhao etal., Scientific Reports (2014) 4:3943] provided a miRNA inhibitionstrategy employing the CRISPR system in murine cells. Zhao usedspecifically designed gRNAs to cut miRNA gene at a single site by Cas9,resulting in knockdown of the miRNA in these cells.

With regard to plant genome editing, Bortesi and Fischer [Bortesi andFischer, Biotechnology Advances (2015) 33: 41-52] discussed the use ofCRISPR-Cas technology in plants as compared to ZFNs and TALENs, andBasak and Nithin [Basak and Nithin, Front Plant Sci. (2015) 6: 1001]teach that CRISPR-Cas technology has been applied for knockdown ofprotein-coding genes in model plants such as Arabidopsis and tobacco andcrops like wheat, maize, and rice.

In addition to disruption of miRNA activity or target binding sites,gene silencing using artificial microRNAs (amiRNAs) mediated genesilencing of endogenous and exogenous target genes were used [Tiwari etal. Plant Mol Biol (2014) 86: 1]. Similar to microRNAs, amiRNAs aresingle-stranded, approximately 21 nucleotides (nt) long, and designed byreplacing the mature miRNA sequences of duplex within pre-miRNAs [Tiwariet al. (2014) supra]. These amiRNAs are introduced as a transgene withinan artificial expression cassette (including a promoter, terminatoretc.) [Carbonell et al., Plant Physiology (2014) pp. 113.234989], areprocessed via small RNA biogenesis and silencing machinery anddownregulate target expression. According to Schwab et al. [Schwab etal. The Plant Cell (2006) Vol. 18, 1121-1133], amiRNAs are active whenexpressed under tissue-specific or inducible promoters and can be usedfor specific gene silencing in plants, especially when several related,but not identical, target genes need to be downregulated.

Senis et al. [Senis et al., Nucleic Acids Research (2017) Vol. 45(1):e3] disclose engineering of a promoterless anti-viral RNAi hairpin intoan endogenous miRNA locus. Specifically, Senis et al. insert an amiRNAprecursor transgene (hairpin pri-amiRNA) adjacent to a naturallyoccurring miRNA gene (e.g. miR122) by homology-directed DNArecombination that is induced by sequence-specific nuclease such as Cas9or TALEN. This approach uses promoter- and terminator-free amiRNAs byutilizing transcriptionally active DNA that expresses natural miRNA(miR122), that is, the endogenous promoter and terminator drove andregulated the transcription of the inserted amiRNA transgene.

Various DNA-free methods of introducing RNA and/or proteins into cellshave been previously described. For example, RNA transfection usingelectroporation and lipofection has been described in U.S. PatentApplication No. 20160289675. Direct delivery of Cas9/gRNAribonucleoprotein (RNP) complexes to cells by microinjection of Cas9protein and gRNA complexes was described by Cho [Cho et al., “Heritablegene knockout in Caenorhabditis elegans by direct injection ofCas9-sgRNA ribonucleoproteins,” Genetics (2013) 195:1177-1180]. Deliveryof Cas9 protein/gRNA complexes via electroporation was described by Kim[Kim et al., “Highly efficient RNA-guided genome editing in human cellsvia delivery of purified Cas9 ribonucleoproteins” Genome Res. (2014)24:1012-1019]. Delivery of Cas9 protein-associated gRNA complexes vialiposomes was reported by Zuris [Zuris et al., “Cationic lipid-mediateddelivery of proteins enables efficient protein-based genome editing invitro and in vivo” Nat Biotechnol. (2014) doi: 10.1038/nbt.3081].

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of modifying a gene encoding or processedinto a non-coding RNA molecule having no RNA silencing activity in aeukaryotic cell, with the proviso that the eukaryotic cell is not aplant cell, the method comprising introducing into the eukaryotic cell aDNA editing agent conferring a silencing specificity of the non-codingRNA molecule towards a target RNA of interest, thereby modifying thegene encoding or processed into the non-coding RNA molecule.

According to an aspect of some embodiments of the present inventionthere is provided a method of modifying a gene encoding or processedinto a non-coding RNA molecule having no RNA silencing activity in aeukaryotic cell, with the proviso that the eukaryotic cell is not aplant cell, the method comprising introducing into the eukaryotic cell aDNA editing agent conferring a silencing specificity of the non-codingRNA molecule towards a target RNA of interest.

According to an aspect of some embodiments of the present inventionthere is provided a method of modifying a gene encoding or processedinto a RNA silencing molecule to a target RNA in a eukaryotic cell, withthe proviso that the eukaryotic cell is not a plant cell, the methodcomprising introducing into the eukaryotic cell a DNA editing agentwhich redirects a silencing specificity of the RNA silencing moleculetowards a second target RNA, the target RNA and the second target RNAbeing distinct, thereby modifying the gene encoding the RNA silencingmolecule.

According to an aspect of some embodiments of the present inventionthere is provided a method of modifying a gene encoding or processedinto a RNA silencing molecule to a target RNA in a eukaryotic cell, withthe proviso that the eukaryotic cell is not a plant cell, the methodcomprising introducing into the eukaryotic cell a DNA editing agentwhich redirects a silencing specificity of the RNA silencing moleculetowards a second target RNA, the target RNA and the second target RNAbeing distinct.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating an infectious disease in asubject in need thereof, the method comprising modifying a gene encodingor processed into a non-coding RNA molecule or encoding or processedinto an RNA silencing molecule according to the method of someembodiments of the invention, wherein the target RNA of interest isassociated with onset or progression of the infectious disease, therebytreating the infectious disease in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a monogenic recessive disorder ina subject in need thereof, the method comprising modifying a geneencoding or processed into a non-coding RNA molecule or encoding orprocessed into an RNA silencing molecule according to the method of someembodiments of the invention, wherein the target RNA of interest isassociated with the monogenic recessive disorder, thereby treating themonogenic recessive disorder in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating an autoimmune disease in asubject in need thereof, the method comprising modifying a gene encodingor processed into a non-coding RNA molecule or encoding or processedinto an RNA silencing molecule according to the method of someembodiments of the invention, wherein the target RNA of interest isassociated with the autoimmune disease, thereby treating the autoimmunedisease in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a cancerous disease in a subjectin need thereof, the method comprising modifying a gene encoding orprocessed into a non-coding RNA molecule or encoding or processed intoan RNA silencing molecule according to the method of some embodiments ofthe invention, wherein the target RNA of interest is associated with thecancerous disease, thereby treating the cancerous disease in thesubject.

According to an aspect of some embodiments of the present inventionthere is provided a method of enhancing efficacy and/or specificity of achemotherapeutic agent in a subject in need thereof, the methodcomprising modifying a gene encoding or processed into a non-coding RNAmolecule or encoding or processed into an RNA silencing moleculeaccording to the method of some embodiments of the invention, whereinthe target RNA of interest is associated with enhancement of efficacyand/or specificity of the chemotherapeutic agent, thereby enhancingefficacy and/or specificity of a chemotherapeutic agent in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of inducing cell apoptosis in a subject inneed thereof, the method comprising modifying a gene encoding orprocessed into a non-coding RNA molecule or encoding or processed intoan RNA silencing molecule according to the method of some embodiments ofthe invention, wherein the target RNA of interest is associated with theapoptosis, thereby inducing cell apoptosis in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a eukaryotic non-humanorganism, with the proviso that the organism is not a plant, wherein atleast some of the cells of the organism comprise a modified geneencoding or processed into a non-coding RNA molecule comprising asilencing specificity towards a target RNA of interest, the methodcomprising modifying a gene according to the method of some embodimentsof the invention, thereby generating the eukaryotic non-human organism.

According to some embodiments of the invention, the gene encoding orprocessed into the non-coding RNA molecule is endogenous to theeukaryotic cell.

According to some embodiments of the invention, the gene encoding theRNA silencing molecule is endogenous to the eukaryotic cell.

According to some embodiments of the invention, modifying the geneencoding or processed into the non-coding RNA molecule comprisesimparting the non-coding RNA molecule with at least 45% complementaritytowards the target RNA of interest.

According to some embodiments of the invention, modifying the geneencoding the RNA silencing molecule comprises imparting the RNAsilencing molecule with at least 45% complementarity towards the secondtarget RNA.

According to some embodiments of the invention, the silencingspecificity of the non-coding RNA molecule is determined by measuring aRNA or protein level of the target RNA of interest.

According to some embodiments of the invention, the silencingspecificity of the RNA silencing molecule is determined by measuring aRNA or protein level of the second target RNA.

According to some embodiments of the invention, the silencingspecificity of the non-coding RNA molecule or the RNA silencing moleculeis determined phenotypically.

According to some embodiments of the invention, determinedphenotypically is effected by determination of at least one phenotypeselected from the group consisting of a cell size, a growthrate/inhibition, a cell shape, a cell membrane integrity, a tumor size,a tumor shape, a pigmentation of an organism, an infection parameter andan inflammation parameter.

According to some embodiments of the invention, the silencingspecificity of the non-coding RNA molecule or the RNA silencing moleculeis determined genotypically.

According to some embodiments of the invention, the phenotype isdetermined prior to a genotype.

According to some embodiments of the invention, the genotype isdetermined prior to a phenotype.

According to some embodiments of the invention, the non-coding RNAmolecule or the RNA silencing molecule is processed from a precursor.

According to some embodiments of the invention, the non-coding RNAmolecule or the RNA silencing molecule is a RNA interference (RNAi)molecule.

According to some embodiments of the invention, the RNAi molecule isselected from the group consisting of a small interfering RNA (siRNA), ashort hairpin RNA (shRNA), a microRNA (miRNA), a Piwi-interacting RNA(piRNA) and trans-acting siRNA (tasiRNA).

According to some embodiments of the invention, the non-coding RNAmolecule is selected from the group consisting of a small nuclear RNA(snRNA), a small nucleolar RNA (snoRNA), a long non-coding RNA (lncRNA),a ribosomal RNA (rRNA), transfer RNA (tRNA), a repeat-derived RNA, and atransposable element RNA.

According to some embodiments of the invention, the RNAi molecule ismodified to preserve originality of structure and to be recognized bycellular RNAi factors.

According to some embodiments of the invention, modifying the gene isaffected by a modification selected from the group consisting of adeletion, an insertion, a point mutation and a combination thereof.

According to some embodiments of the invention, the modification is in astem region of the non-coding RNA molecule or the RNA silencingmolecule.

According to some embodiments of the invention, the modification is in aloop region of the non-coding RNA molecule or the RNA silencingmolecule.

According to some embodiments of the invention, the modification is in anon-structured region of the non-coding RNA molecule or the RNAsilencing molecule.

According to some embodiments of the invention, the modification is in astem region and a loop region of the non-coding RNA molecule or the RNAsilencing molecule.

According to some embodiments of the invention, the modification is in astem region and a loop region and in non-structured region of thenon-coding RNA molecule or the RNA silencing molecule.

According to some embodiments of the invention, the modification is aninsertion.

According to some embodiments of the invention, the modification is adeletion.

According to some embodiments of the invention, the modification is apoint mutation.

According to some embodiments of the invention, the modificationcomprises a modification of at most 200 nucleotides.

According to some embodiments of the invention, the method furthercomprises introducing into the eukaryotic cell donor oligonucleotides.

According to some embodiments of the invention, the DNA editing agentcomprises at least one gRNA operatively linked to a plant expressiblepromoter.

According to some embodiments of the invention, the DNA editing agentdoes not comprise an endonuclease.

According to some embodiments of the invention, the DNA editing agentcomprises an endonuclease.

According to some embodiments of the invention, the DNA editing agentcomprises a DNA editing system selected from the group consisting of ameganuclease, a zinc finger nucleases (ZFN), a transcription-activatorlike effector nuclease (TALEN) and CRISPR.

According to some embodiments of the invention, the endonucleasecomprises Cas9.

According to some embodiments of the invention, the DNA editing agent isapplied to the cell as DNA, RNA or RNP.

According to some embodiments of the invention, the DNA editing agent islinked to a reporter for monitoring expression in a eukaryotic cell.

According to some embodiments of the invention, the reporter is afluorescent protein.

According to some embodiments of the invention, the target RNA ofinterest or the second target RNA is endogenous to the eukaryotic cell.

According to some embodiments of the invention, the target RNA ofinterest or the second target RNA is associated with a cancer.

According to some embodiments of the invention, the target RNA ofinterest or the second target RNA is exogenous to the eukaryotic cell.

According to some embodiments of the invention, the target RNA ofinterest or the second target RNA is associated with an infectiousdisease.

According to some embodiments of the invention, the eukaryotic cell isobtained from a eukaryotic organism selected from the group consistingof a mammal, an insect, a nematode, a bird, a reptile, a fish, acrustacean, a fungi and an algae.

According to some embodiments of the invention, the eukaryotic cell is amammalian cell.

According to some embodiments of the invention, the mammalian cellcomprises a human cell.

According to some embodiments of the invention, the eukaryotic cell is atotipotent stem cell.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a flow chart of an embodiment computational pipeline togenerate Genome Editing Induced Gene Silencing (GEiGS) templates. Thecomputational GEiGS pipeline applies biological metadata and enables anautomatic generation of GEiGS DNA templates that are used to minimallyedit miRNA genes, leading to a new gain of function, i.e. redirection oftheir silencing capacity to target sequence of interest.

FIG. 2 is an embodiment flowchart of GEiGS replacement of miRNA withsiRNA targeting Green Fluorescent Protein (GFP), generating silencing ofthe stably expressed GFP gene in human cell lines.

FIGS. 3A-B are photographs illustrating knock down of GFP expressionlevels in human cells. Control cells (FIG. 3A) stably express GFP athigh levels as compared to cells stably expressing siGFP in which GFPexpression is silenced (FIG. 3B).

FIG. 4 is an embodiment flowchart of GEiGS cells stably expressingsiGFP. All positive transfection events are red fluorescent proteins(RFP)+GFP. However, since GEiGS cells stably express siGFP, positivetransfected cells show only red fluorescent expression.

FIG. 5 is an embodiment flowchart of GEiGS cells stably expressing siRNAtargeting p53. All positive transfection events are GFP and evadechemotherapy or the hDM2 inhibitor Nutlin3-induced cell death.

FIG. 6 is an embodiment flowchart of GEiGS cells stably expressing siRNAtargeting pro-apoptotic genes in human cancer cell line U2OS. Allpositive transfection events are RFP and evade chemotherapy-induced celldeath.

FIG. 7 is an embodiment flowchart of GEiGS cells generated resistant tolentivirus infection (GFP is used as the virus marker gene or as theexogenous gene).

FIG. 8 is an embodiment flowchart of GEiGS cells generated resistant tovirus infection (i.e. immunization of cells towards an exogenous viralgene).

FIG. 9 is an embodiment drawing illustrating the main stages required todesign RNA silencing molecule and with minimally edited miRNA genebases.

FIG. 10 is a graph illustrating the diverse non-coding RNA types thatare actively engaged in RNA interference (RNAi). The list providesnon-coding RNA types that are both Dicer substrates (proven to be boundby Dicer) and are processed into small silencing RNA (their small RNAsare proven to be bound by Argonaute proteins) (axis y). Each type hasmultiple slightly different subtypes (axis x).

FIGS. 11A-E is an embodiment example of human non-coding RNAs that showthe non-coding RNA precursor and its derived Ago-bound small RNAs. Shownare the AGO2- and AGO3-bound small RNAs mapped to Dicer-bound non-codingRNAs precursors. (FIG. 11A) shows the let7 microRNA and its primary(marked in blue line) and secondary mature miRNA sites (represented bygray bars). (FIGS. 11B-E) show examples of other biotypes where thesmall RNA mapping shows a signature analog to the one found inmicroRNAs.

FIGS. 12A-E are embodiment examples of GEiGS oligo designs. Theselections of non-coding RNA precursors that give rise to mature smallRNA molecules are highlighted in green. Sequence differences between theGEiGS oligos and the wild type sequence are highlighted in red. (FIG.12A) Embodiment examples of GEiGS oligo designs in which the GEiGSprecursors preserve identical secondary structure as the wild-type (wt)non-coding RNA. Design based on the Human microRNA-100. From left toright: wild-type microRNA, GEiGS design with matching structure andminimal sequence changes, and GEiGS design with matching structure andmaximal sequence changes. Of note, the GeiGS designs were based on 21ntsiRNAs targeting Human heparin-binding vascular endothelial growthfactor (VEGF); (FIG. 12B) Embodiment examples of GEiGS oligo designs inwhich the GEiGS precursors do not preserve the secondary structure asthe wt non-coding RNA. Design based on the Human microRNA-100. From leftto right: wild-type microRNA, GEiGS design with non-matching structureand minimal sequence changes, and GEiGS design with non-matchingstructure and maximal sequence changes. Of note, the GeiGS designs werebased on 21nt siRNAs targeting Human heparin-binding vascularendothelial growth factor (VEGF); (FIG. 12C) Embodiment examples ofGEiGS oligo designs in which the GEiGS precursors preserve identicalsecondary structure as the wt non-coding RNA. Design based on theCID_001033 tRNA. From left to right: wild-type tRNA, GEiGS design withmatching structure and minimal sequence changes, and GEiGS design withmatching structure and maximal sequence changes. Of note, the GeiGSdesigns were based on 21nt siRNAs targeting the bcr/abl e8a2 fusionprotein gene; (FIG. 12D) Embodiment examples of GEiGS oligo designs inwhich the GEiGS precursors do not preserve the secondary structure asthe wt non-coding RNA. Design based on the CID_001033 tRNA. From left toright, wild-type tRNA, GEiGS design with non-matching structure andminimal sequence changes, and GEiGS design with non-matching structureand maximal sequence changes. The GEiGS designs were based on 21ntsiRNAS targeting the bcr/abl e8a2 fusion protein gene; (FIG. 12E)Embodiment examples of GEiGS oligo designs in which the precursorstructure does not play a role in the biogenesis, hence, it is notrequired to be maintained. Design based on the Brassica rapa bnTAS3BtasiRNA. From left to right: wild-type tasiRNA, GEiGS design withminimal sequence changes, and GEiGS design with maximal sequencechanges. Of note, the circular structure is not inherent to the moleculeand was applied for convenience; tasiRNA biogenesis, unlike miRNAs andtRNAs, does not rely on the precursor secondary structure (as discussedin detail in Borges and Martienssen (2015) Nature Reviews Molecular CellBiology | AOP, published online 4 Nov. 2015; doi:10.1038/nrm4085). Belowthe full molecules there is a detail of the section containingmodifications. The GEiGS designs were based on 21nt siRNAS targeting thebcr/abl e8a2 fusion protein gene;

FIG. 13 illustrates PDS3 Phenotype/Genotype: bleached phenotype plantswere selected and genotyped through internal amplicon PCR followed byrestriction digest analysis with BtsαI (NEB) in order to verify donorpresence vs. wild type sequence. Lane 1: Treated plants with NO DONOR,restricted, Lanes 2-4: PDS3 treated plants containing DONOR restricted,Lane 5: Positive plasmid DONOR control unrestricted, Lane 6: Water notemplate control, Lane 7: Positive Plasmid DONOR restricted, Lane 8:Plants bombarded with negative DONOR restricted, Lane 9: Untreatedcontrol plants restricted. Subsequent external PCR amplification of theamplicon was processed and sequenced in order to validate the insertion.

FIG. 14 illustrates ADH1 Phenotype/Genotype: Plants were selectedthrough Allyl alcohol resistance and genotyped through internal ampliconPCR followed by Bccl (NEB) restriction digest in order to verify donorpresence. Lane 1: Allyl alcohol sensitive control plant restricted, Lane2-4: Allyl alcohol resistant plants containing DONOR restricted, Lane5:Positive plasmid DONOR control unrestricted, Lane 6: no templatecontrol, Lane7: Positive Plasmid DONOR restricted, Lane 8: Plantbombarded with non-specific DONOR restricted, Lane 9: Non Allyl alcoholtreated control restricted.

FIG. 15 is a graph illustrating gene expression analysis in miR-173modified plant targeting AtPDS3 transcript. Analysis of AtPDS3expression was carried out through qRT-PCR, in regenerating bombardedplants with GEiGS #4 and SWAP3 compared to plants bombarded with GEiGS#5 and SWAP1 and 2 (GFP). Of note, a reduction of 82% in gene expressionlevel, on the average, was observed, when miR-173 was modified to targetAtPDS3, compared to control plants (Error bars present SD; p-value <0.01calculated on Ct values).

FIG. 16 is a graph illustrating gene expression analysis in miR-390modified plant targeting AtPDS3 transcript. Analysis of AtADH1expression was carried out through qRT-PCR, in regenerating bombardedplants with GEiGS #1 and SWAP11, compared to plants bombarded with GEiGS#5 and SWAP1 and 2 (GFP). Of note, a reduction of 82% in gene expressionlevel, on the average, was observed, when miR-390 was modified to targetAtADH1, compared to control plants (Error bars represent SD; p-value<0.01 calculated on Ct values).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to modifyinggenes that encode or are processed into non-coding RNA molecules,including RNA silencing molecules and, more particularly, but notexclusively, to the use of same for silencing endogenous or exogenoustarget RNA of interest in eukaryotic cells which are not plant cells.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

Two of the most powerful genetic therapeutic technologies developed thusfar are gene therapy, which enables restoration of missing gene functionby viral transgene expression, and RNAi, which mediates repression ofdefective genes by knockdown of the target mRNA. Recent advances ingenome editing techniques have also made it possible to alter DNAsequences in living cells by editing a few nucleotides in cells of humanpatients such as by genome editing (NHEJ and HR) following induction ofsite-specific double-strand breaks (DSBs) at desired locations in thegenome.

While reducing the present invention to practice, the present inventorshave devised a gene editing technology utilizing non-coding RNAmolecules designed to target and interfere with any target gene ofinterest (endogenous or exogenous to the eukaryotic cell). The geneediting technology described herein does not necessitate the classicalmolecular genetic and transgenic tools comprising expression cassettesthat have a promoter, terminator, selection marker. Moreover, the geneediting technology of some embodiments of the invention comprises genomeediting of a non-coding RNA molecule (e.g. endogenous) yet it is stableand heritable.

As is shown herein below and in the Examples section which follows, thepresent inventors have designed a Genome Editing Induced Gene Silencing(GEiGS) platform capable of utilizing a eukaryotic cell's endogenousnon-coding RNA molecules including e.g. RNA silencing molecules (e.g.siRNA, miRNA, piRNA, tasiRNA, tRNA, rRNA, antisense RNA, etc.) andmodifying them to target any RNA target of interest (see exemplaryflowchart in FIG. 2). Using GEiGS, the present method enables screeningof potential non-coding RNA molecules, editing a few nucleotides inthese endogenous RNA molecules, and thereby redirecting their activityand/or specificity to effectively and specifically target any RNA ofinterest including, for instance, endogenous RNA coding for mutatedproteins (e.g. oncogenes in cancers) or exogenous RNA encoded bypathogens (see exemplary flowchart in FIG. 1). Taken together, GEiGS canbe utilized as a novel technology for modulation of endogenous geneexpression and also to immunize organisms to different biotic andabiotic stresses such as e.g. cancer, viruses, insects, fungi,nematodes, heat, drought, starvation etc.

Thus, according to one aspect of the present invention there is provideda method of modifying a gene encoding or processed into a non-coding RNAmolecule having no RNA silencing activity in a eukaryotic cell, with theproviso that the eukaryotic cell is not a plant cell, the methodcomprising introducing into the eukaryotic cell a DNA editing agentconferring a silencing specificity of the non-coding RNA moleculetowards a target RNA of interest, thereby modifying the gene encoding orprocessed into the non-coding RNA molecule.

According to another aspect of the invention there is provided a methodof modifying a gene encoding or processed into a RNA silencing moleculeto a target RNA in a eukaryotic cell, with the proviso that theeukaryotic cell is not a plant cell, the method comprising introducinginto the eukaryotic cell a DNA editing agent which redirects a silencingspecificity of the RNA silencing molecule towards a second target RNA,the target RNA and the second target RNA being distinct, therebymodifying the gene encoding the RNA silencing molecule.

The term “eukaryotic cell” as used herein refers to any cell of aeukaryotic organism. Eukaryotic organisms include single- andmulti-cellular organisms. Single cell eukaryotic organisms include, butare not limited to, yeast, protozoans, slime molds and algae.Multi-cellular eukaryotic organisms include, but are not limited to,animals (e.g. mammals, insects, nematodes, birds, fish, reptiles andcrustaceans), fungi and algae (e.g. brown algae, red algae, greenalgae).

According to one embodiment, the eukaryotic cell is not a cell of aplant.

According to a one embodiment, the eukaryotic cell is an animal cell.

According to a one embodiment, the eukaryotic cell is a cell of avertebrate.

According to a one embodiment, the eukaryotic cell is a cell of aninvertebrate.

According to a specific embodiment, the invertebrate cell is a cell ofan insect, a snail, a clam, an octopus, a starfish, a sea-urchin, ajellyfish, and a worm.

According to a specific embodiment, the invertebrate cell is a cell of acrustacean. Exemplary crustaceans include, but are not limited to,shrimp, prawns, crabs, lobsters and crayfishes.

According to a specific embodiment, the invertebrate cell is a cell of afish. Exemplary fish include, but are not limited to, Salmon, Tuna,Pollock, Catfish, Cod, Haddock, Prawns, Sea bass, Tilapia, Arctic charand Carp.

According to a one embodiment, the eukaryotic cell is a mammalian cell.

According to a specific embodiment, the mammalian cell is a cell of anon-human organism, such as but not limited to, a rodent, a rabbit, apig, a goat, a ruminant (e.g. cattle, sheep, antelope, deer, andgiraffe), a dog, a cat, a horse, and non-human primate.

According to a specific embodiment, the eukaryotic cell is a cell ofhuman being.

According to one embodiment, the eukaryotic cell is a primary cell, acell line, a somatic cell, a germ cell, a stem cell, an embryonic stemcell, an adult stem cell, a hematopoietic stem cell, a mesenchymal stemcell, an induced pluripotent stem cell (iPS), a gamete cell, a zygotecell, a blastocyst cell, an embryo, a fetus and/or a donor cell.

As used herein, the phrase “stem cells” refers to cells which arecapable of remaining in an undifferentiated state (e.g., totipotent,pluripotent or multipotent stem cells) for extended periods of time inculture until induced to differentiate into other cell types having aparticular, specialized function (e.g., fully differentiated cells).Totipotent cells, such as embryonic cells within the first couple ofcell divisions after fertilization are the only cells that candifferentiate into embryonic and extra-embryonic cells and are able todevelop into a viable human being. Preferably, the phrase “pluripotentstem cells” refers to cells which can differentiate into all threeembryonic germ layers, i.e., ectoderm, endoderm and mesoderm orremaining in an undifferentiated state. The pluripotent stem cellsinclude embryonic stem cells (ESCs) and induced pluripotent stem cells(iPS). The multipotent stem cells include adult stem cells andhematopoietic stem cells.

The phrase “embryonic stem cells” refers to embryonic cells which arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation of the embryo (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeWO2006/040763), embryonic germ (EG) cells which are obtained from thegenital tissue of a fetus any time during gestation, preferably before10 weeks of gestation, and cells originating from an unfertilized ovawhich are stimulated by parthenogenesis (parthenotes).

The embryonic stem cells of some embodiments of the invention can beobtained using well-known cell-culture methods. For example, humanembryonic stem cells can be isolated from human blastocysts. Humanblastocysts are typically obtained from human in vivo preimplantationembryos or from in vitro fertilized (IVF) embryos. Alternatively, asingle cell human embryo can be expanded to the blastocyst stage.

It will be appreciated that commercially available stem cells can alsobe used according to some embodiments of the invention. Human ES cellscan be purchased from the NIH human embryonic stem cells registry[www(dot)grants (dot) nih (dot) gov/stem_cells/registry/current (dot)htm].

In addition, embryonic stem cells can be obtained from various species,including mouse (Mills and Bradley, 2001), golden hamster [Doetschman etal., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, DevBiol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesusmonkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

“Induced pluripotent stem cells” (iPS; embryonic-like stem cells) refersto cells obtained by de-differentiation of adult somatic cells which areendowed with pluripotency (i.e., being capable of differentiating intothe three embryonic germ cell layers, i.e., endoderm, ectoderm andmesoderm). According to some embodiments of the invention, such cellsare obtained from a differentiated tissue (e.g., a somatic tissue suchas skin) and undergo de-differentiation by genetic manipulation whichreprogram the cell to acquire embryonic stem cells characteristics.According to some embodiments of the invention, the induced pluripotentstem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4and c-Myc in a somatic stem cell.

Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can begenerated from somatic cells by genetic manipulation of somatic cells,e.g., by retroviral transduction of somatic cells such as fibroblasts,hepatocytes, gastric epithelial cells with transcription factors such asOct-3/4, Sox2, c-Myc, and KLF4 [such as described in Park et al.Reprogramming of human somatic cells to pluripotency with definedfactors. Nature (2008) 451:141-146].

The phrase “adult stem cells” (also called “tissue stem cells” or a stemcell from a somatic tissue) refers to any stem cell derived from asomatic tissue [of either a postnatal or prenatal animal (especially thehuman)]. The adult stem cell is generally thought to be a multipotentstem cell, capable of differentiation into multiple cell types. Adultstem cells can be derived from any adult, neonatal or fetal tissue suchas adipose tissue, skin, kidney, liver, prostate, pancreas, intestine,bone marrow and placenta.

According to one embodiment, the stem cells utilized by some embodimentsof the invention are bone marrow (BM)-derived stem cells includinghematopoietic, stromal or mesenchymal stem cells [Dominici, M et al.,(2001) J. Biol. Regul. Homeost. Agents. 15: 28-37]. BM-derived stemcells may be obtained from iliac crest, femora, tibiae, spine, rib orother medullar spaces.

Hematopoietic stem cells (HSCs), which may also referred to as adulttissue stem cells, include stem cells obtained from blood or bone marrowtissue of an individual at any age or from cord blood of a newbornindividual. Preferred stem cells according to this aspect of someembodiments of the invention are embryonic stem cells, preferably of ahuman or primate (e.g., monkey) origin.

Placental and cord blood stem cells may also be referred to as “youngstem cells”.

Mesenchymal stem cells (MSCs), the formative pluripotent blast cells,give rise to one or more mesenchymal tissues (e.g., adipose, osseous,cartilaginous, elastic and fibrous connective tissues, myoblasts) aswell as to tissues other than those originating in the embryonicmesoderm (e.g., neural cells) depending upon various influences frombioactive factors such as cytokines. Although such cells can be isolatedfrom embryonic yolk sac, placenta, umbilical cord, fetal and adolescentskin, blood and other tissues, their abundance in the BM far exceedstheir abundance in other tissues and as such isolation from BM ispresently preferred.

Adult tissue stem cells can be isolated using various methods known inthe art such as those disclosed by Alison, M. R. [J Pathol. (2003)200(5): 547-50]. Fetal stem cells can be isolated using various methodsknown in the art such as those disclosed by Eventov-Friedman S, et al.[PLoS Med. (2006) 3: e215].

Hematopoietic stem cells can be isolated using various methods known inthe arts such as those disclosed by “Handbook of Stem Cells” edit byRobert Lanze, Elsevier Academic Press, 2004, Chapter 54, pp609-614,isolation and characterization of hematopoietic stem cells, by Gerald JSpangrude and William B Stayton.

Methods of isolating, purifying and expanding mesenchymal stem cells(MSCs) are known in the arts and include, for example, those disclosedby Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and Jones E. A. etal., 2002, Isolation and characterization of bone marrow multipotentialmesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

According to one embodiment, the eukaryotic cell is isolated from itsnatural environment (e.g. human body).

According to one embodiment, the eukaryotic cell is a healthy cell.

According to one embodiment, the eukaryotic cell is a diseased cell or acell prone to a disease.

According to one embodiment, the eukaryotic cell is a cancer cell.

According to one embodiment, the eukaryotic cell is an immune cell (e.g.T cell, B cell, macrophage, NK cell, etc.).

According to one embodiment, the eukaryotic cell is a cell infected by apathogen (e.g. by a bacterial, viral or fungal pathogen).

As used herein, the term “non-coding RNA molecule” refers to a RNAsequence that is not translated into an amino acid sequence and does notencode a protein.

According to one embodiment, the non-coding RNA molecule is typicallysubject to the RNA silencing processing mechanism or activity. However,also contemplated herein are a few changes in nucleotides (e.g. up to 24nucleotides) which may elicit a processing mechanism that results in RNAinterference or translation inhibition.

According to a specific embodiment, the non-coding RNA molecule isendogenous (naturally occurring, e.g. native) to the cell.

It will be appreciated that the non-coding RNA molecule can also beexogenous to the cell (i.e. externally added and which is not naturallyoccurring in the cell).

According to some embodiments, the non-coding RNA molecule comprises anintrinsic translational inhibition activity.

According to some embodiments, the non-coding RNA molecule comprises anintrinsic RNAi activity.

According to some embodiments, the non-coding RNA molecule does notcomprise an intrinsic translational inhibition activity or an intrinsicRNAi activity (i.e. the non-coding RNA molecule does not have an RNAsilencing activity).

According to an embodiment of the invention, the non-coding RNA moleculeis specific to a target RNA (e.g., a natural target RNA) and does notcross inhibit or silence a second target RNA or target RNA of interestunless designed to do so (as discussed below) exhibiting 100% or lessglobal homology to the target gene, e.g., less than 99%, less than 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%,83%, 82%, 81% global homology to the target gene; as determined at theRNA or protein level by RT-PCR, Western blot, Immunohistochemistryand/or flow cytometry or any other detection methods.

According to one embodiment, the non-coding RNA molecule is a RNAsilencing or RNA interference (RNAi) molecule.

The term “RNA silencing” or RNAi refers to a cellular regulatorymechanism in which non-coding RNA molecules (the “RNA silencingmolecule” or “RNAi molecule”) mediate, in a sequence specific manner,co- or post-transcriptional inhibition of gene expression ortranslation.

According to one embodiment, the RNA silencing molecule is capable ofmediating RNA repression during transcription (co-transcriptional genesilencing).

According to a specific embodiment, co-transcriptional gene silencingincludes epigenetic silencing (e.g. chromatic state that prevents geneexpression).

According to one embodiment, the RNA silencing molecule is capable ofmediating RNA repression after transcription (post-transcriptional genesilencing).

Post-transcriptional genes silencing (PTGS) typically refers to theprocess of degradation or cleavage of messenger RNA (mRNA) moleculeswhich decrease their activity by preventing translation. For example,and as discussed in detail below, a guide strand of a RNA silencingmolecule pairs with a complementary sequence in a mRNA molecule andinduces cleavage by e.g. Argonaute 2 (Ago2).

Co-transcriptional gene silencing typically refers to inactivation ofgene activity (i.e. transcription repression) and typically occurs inthe cell nucleus. Such gene activity repression is mediated byepigenetic-related factors, such as e.g. methyl-transferases, thatmethylate target DNA and histones. Thus, in co-transcriptional genesilencing, the association of a small RNA with a target RNA (smallRNA-transcript interaction) destabilizes the target nascent transcriptand recruits DNA- and histone-modifying enzymes (i.e. epigeneticfactors) that induce chromatin remodeling into a structure that repressgene activity and transcription. Also, in co-transcriptional genesilencing, chromatin-associated long non-coding RNA scaffolds mayrecruit chromatin-modifying complexes independently of small RNAs. Theseco-transcriptional silencing mechanisms form RNA surveillance systemsthat detect and silence inappropriate transcription events, and providea memory of these events via self-reinforcing epigenetic loops [asdescribed in D. Hoch and D. Moazed, RNA-mediated epigenetic regulationof gene expression, Nat Rev Genet. (2015) 16(2): 71-84].

According to an embodiment of the invention, the RNAibiogenesis/processing machinery generates the RNA silencing molecule.

According to an embodiment of the invention, the RNAibiogenesis/processing machinery generates the RNA silencing molecule,but no specific target has been identified.

According to one embodiment, the non-coding RNA molecule is a capable ofinducing RNA interference (RNAi).

Following is a detailed description of non-coding RNA molecules whichcomprise an intrinsic RNAi activity (e.g. are RNA silencing molecules)that can be used according to specific embodiments of the presentinvention.

According to one embodiment, the non-coding RNA molecule or the RNAsilencing molecule is processed from a precursor.

According to one embodiment, the non-coding RNA molecule or RNAsilencing molecule is processed from a single stranded RNA (ssRNA)precursor.

According to one embodiment, the non-coding RNA molecule or the RNAsilencing molecule is processed from a duplex-structured single-strandedRNA precursor.

According to one embodiment, the non-coding RNA molecule or RNAsilencing molecule is processed from a dsRNA precursor (e.g. comprisingperfect and imperfect base pairing).

According to one embodiment, the non-coding RNA molecule or the RNAsilencing molecule is processed from a non-structured RNA precursor.

According to one embodiment, the non-coding RNA molecule or the RNAsilencing molecule is processed from a protein-coding RNA precursor.

According to one embodiment, the non-coding RNA molecule or the RNAsilencing molecule is processed from a non-coding RNA precursor.

According to one embodiment, the dsRNA can be derived from two differentcomplementary RNAs, or from a single RNA that folds on itself to formdsRNA.

Perfect and imperfect based paired RNA (i.e. double stranded RNA,dsRNA), siRNA and shRNA—The presence of long dsRNAs in cells stimulatesthe activity of a ribonuclease III enzyme referred to as dicer. Dicer,also known as endoribonuclease Dicer or helicase with RNase motif, is anenzyme that in humans is encoded by the DICER1 gene. Dicer is involvedin the processing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs). siRNAs derived from dicer activity aretypically about 21 to about 23 nucleotides in length and comprise about19 base pair duplexes with two 3′ nucleotides overhangs.

Accordingly, some embodiments of the invention contemplate modifying agene encoding a dsRNA to redirect a silencing specificity (includingsilencing activity) towards a second target RNA (i.e. RNA of interest).

According to one embodiment dsRNA precursors longer than 21 bp are used.Various studies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example [Strat et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 18-30 base pairs) that induce the RNA interference (RNAi)pathway. Typically, siRNAs are chemically synthesized as 21 mers with acentral 19 bp duplex region and symmetric 2-base 3′-overhangs on thetermini, although it has been recently described that chemicallysynthesized RNA duplexes of 25-30 base length can have as much as a100-fold increase in potency compared with 21 mers at the same location.The observed increased potency obtained using longer RNAs in triggeringRNAi is suggested to result from providing Dicer with a substrate(27mer) instead of a product (21mer) and that this improves the rate orefficiency of entry of the siRNA duplex into RISC.

It has been found that position, but not the composition, of the3′-overhang influences potency of a siRNA and asymmetric duplexes havinga 3′-overhang on the antisense strand are generally more potent thanthose with the 3′-overhang on the sense strand (Rose et al., 2005).

The strands of a double-stranded interfering RNA (e.g., a siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., a shRNA).Thus, as mentioned, the RNA silencing molecule of some embodiments ofthe invention may also be a short hairpin RNA (shRNA).

The term short hairpin RNA, “shRNA”, as used herein, refers to a RNAmolecule having a stem-loop structure, comprising a first and secondregion of complementary sequence, the degree of complementarity andorientation of the regions being sufficient such that base pairingoccurs between the regions, the first and second regions being joined bya loop region, the loop resulting from a lack of base pairing betweennucleotides (or nucleotide analogs) within the loop region. The numberof nucleotides in the loop is a number between and including 3 to 23, or5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides inthe loop can be involved in base-pair interactions with othernucleotides in the loop. Examples of oligonucleotide sequences that canbe used to form the loop include 5′-CAAGAGA-3′ and 5′-UUACAA-3′(International Patent Application Nos. WO2013126963 and WO2014107763).It will be recognized by one of skill in the art that the resultingsingle chain oligonucleotide forms a stem-loop or hairpin structurecomprising a double-stranded region capable of interacting with the RNAimachinery.

The RNA silencing molecule of some embodiments of the invention need notbe limited to those molecules containing only RNA, but furtherencompasses chemically-modified nucleotides and non-nucleotides.

Various types of siRNAs are contemplated by the present invention,including trans-acting siRNAs (Ta-siRNAs), repeat-associated siRNAs(Ra-siRNAs) and natural-antisense transcript-derived siRNAs(Nat-siRNAs).

According to one embodiment, silencing RNA includes “piRNA” which is aclass of Piwi-interacting RNAs of about 26 and 31 nucleotides in length.piRNAs typically form RNA-protein complexes through interactions withPiwi proteins, i.e. antisense piRNAs are typically loaded into Piwiproteins (e.g. Piwi, Ago3 and Aubergine (Aub)).

miRNA—According to another embodiment the RNA silencing molecule may bea miRNA.

The term “microRNA”, “miRNA”, and “miR” are synonymous and refer to acollection of non-coding single-stranded RNA molecules of about 19-28nucleotides in length, which regulate gene expression. miRNAs are foundin a wide range of organisms, including viruses, and have been shown toplay a role in development, homeostasis, and disease etiology.

Initially the pre-miRNA is present as a long non-perfect double-strandedstem loop RNA that is further processed by Dicer into a siRNA-likeduplex, comprising the mature guide strand (miRNA) and a similar-sizedfragment known as the passenger strand (miRNA*). The miRNA and miRNA*may be derived from opposing arms of the pri-miRNA and pre-miRNA. miRNA*sequences may be found in libraries of cloned miRNAs but typically atlower frequency than the miRNAs.

Although initially present as a double-stranded species with miRNA*, themiRNA eventually becomes incorporated as a single-stranded RNA into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC). Various proteins can form the RISC, which can lead tovariability in specificity for miRNA/miRNA* duplexes, binding site ofthe target gene, activity of miRNA (repress or activate), and whichstrand of the miRNA/miRNA* duplex is loaded in to the RISC.

When the miRNA strand of the miRNA:miRNA* duplex is loaded into theRISC, the miRNA* is removed and degraded. The strand of the miRNA:miRNA*duplex that is loaded into the RISC is the strand whose 5′ end is lesstightly paired. In cases where both ends of the miRNA:miRNA* haveroughly equivalent 5′ pairing, both miRNA and miRNA* may have genesilencing activity.

The RISC identifies target nucleic acids based on high levels ofcomplementarity between the miRNA and the mRNA, especially bynucleotides 2-8 of the miRNA (referred as “seed sequence”).

A number of studies have looked at the base-pairing requirement betweenmiRNA and its mRNA target for achieving efficient inhibition oftranslation (reviewed by Bartel 2004, Cell 116-281). Computationalstudies, analyzing miRNA binding on whole genomes have suggested aspecific role for bases 2-8 at the 5′ of the miRNA (also referred to as“seed sequence”) in target binding but the role of the first nucleotide,found usually to be “A” was also recognized (Lewis et al 2005 Cell120-15). Similarly, nucleotides 1-7 or 2-8 were used to identify andvalidate targets by Krek et al. (2005, Nat Genet 37-495). The targetsites in the mRNA may be in the 5′ UTR, the 3′ UTR or in the codingregion. Interestingly, multiple miRNAs may regulate the same mRNA targetby recognizing the same or multiple sites. The presence of multiplemiRNA binding sites in most genetically identified targets may indicatethat the cooperative action of multiple RISCs provides the mostefficient translational inhibition.

miRNAs may direct the RISC to downregulate gene expression by either oftwo mechanisms: mRNA cleavage or translational repression. The miRNA mayspecify cleavage of the mRNA if the mRNA has a certain degree ofcomplementarity to the miRNA. When a miRNA guides cleavage, the cut istypically between the nucleotides pairing to residues 10 and 11 of themiRNA. Alternatively, the miRNA may repress translation if the miRNAdoes not have the requisite degree of complementarity to the miRNA.Translational repression may be more prevalent in animals since animalsmay have a lower degree of complementarity between the miRNA and bindingsite.

It should be noted that there may be variability in the 5′ and 3′ endsof any pair of miRNA and miRNA*. This variability may be due tovariability in the enzymatic processing of Drosha and Dicer with respectto the site of cleavage. Variability at the 5′ and 3′ ends of miRNA andmiRNA* may also be due to mismatches in the stem structures of thepri-miRNA and pre-miRNA. The mismatches of the stem strands may lead toa population of different hairpin structures. Variability in the stemstructures may also lead to variability in the products of cleavage byDrosha and Dicer.

It will be appreciated that the pre-miRNA sequence may comprise from45-90, 60-80 or 60-70 nucleotides while the pri-miRNA sequence maycomprise from 45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or 80-100nucleotides.

According to one embodiment, the miRNA comprises miR-150 (e.g. humanmiR-150, e.g. as set forth in SEQ ID NO: 13).

According to one embodiment, the miRNA comprises miR-210 (e.g. humanmiR-210, e.g. as set forth in SEQ ID NO: 14).

According to one embodiment, the miRNA comprises Let-7 (e.g. humanLet-7, e.g. as set forth in SEQ ID NO: 15).

According to one embodiment, the miRNA comprises miR-184 (e.g. humanmiR-184, e.g. as set forth in SEQ ID NO: 16).

According to one embodiment, the miRNA comprises miR-204 (e.g. humanmiR-204, e.g. as set forth in SEQ ID NO: 17).

According to one embodiment, the miRNA comprises miR-25 (e.g. humanmiR-25, e.g. as set forth in SEQ ID NO: 18).

According to one embodiment, the miRNA comprises miR-34 (e.g. humanmiR-34a/b/c, e.g. as set forth in SEQ ID NOs: 19-21, respectively).

Additional miRNAs are provided in Table 1B, hereinbelow.

Antisense—Antisense is a single stranded RNA designed to prevent orinhibit expression of a gene by specifically hybridizing to its mRNA.Downregulation of a target RNA can be effected using an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding the target RNA.

As mentioned, the non-coding RNA molecule may not comprise a canonical(intrinsic) RNAi activity (e.g. is not a canonical RNA silencingmolecule, or its target has not been identified). Such non-coding RNAmolecules include the following:

According to one embodiment, the non-coding RNA molecule is a transferRNA (tRNA). The term “tRNA” refers to a RNA molecule that serves as thephysical link between nucleotide sequence of nucleic acids and the aminoacid sequence of proteins, formerly referred to as soluble RNA or sRNA.tRNA is typically about 76 to 90 nucleotides in length.

According to one embodiment, the non-coding RNA molecule is a ribosomalRNA (rRNA). The term “rRNA” refers to the RNA component of the ribosomei.e. of either the small ribosomal subunit or the large ribosomalsubunit.

According to one embodiment, the non-coding RNA molecule is a smallnuclear RNA (snRNA or U-RNA). The terms “sRNA” or “U-RNA” refer to thesmall RNA molecules found within the splicing speckles and Cajal bodiesof the cell nucleus in eukaryotic cells. snRNA is typically about 150nucleotides in length.

According to one embodiment, the non-coding RNA molecule is a smallnucleolar RNA (snoRNA). The term “snoRNA” refers to the class of smallRNA molecules that primarily guide chemical modifications of other RNAs,e.g. rRNAs, tRNAs and snRNAs. snoRNA is typically classified into one oftwo classes: the C/D box snoRNAs are typically about 70-120 nucleotidesin length and are associated with methylation, and the H/ACA box snoRNAsare typically about 100-200 nucleotides in length and are associatedwith pseudouridylation.

Similar to snoRNAs are the scaRNAs (i.e. Small Cajal body RNA genes)which perform a similar role in RNA maturation to snoRNAs, but theirtargets are spliceosomal snRNAs and they perform site-specificmodifications of spliceosomal snRNA precursors (in the Cajal bodies ofthe nucleus).

According to one embodiment, the non-coding RNA molecule is anextracellular RNA (exRNA). The term “exRNA” refers to RNA speciespresent outside of the cells from which they were transcribed (e.g.exosomal RNA).

According to one embodiment, the non-coding RNA molecule is a longnon-coding RNA (lncRNA). The term “lncRNA” or “long ncRNA” refers tonon-protein coding transcripts typically longer than 200 nucleotides.

According to one embodiment, non-limiting examples of non-coding RNAmolecules include, but are not limited to, microRNA (miRNA),piwi-interacting RNA (piRNA), short interfering RNA (siRNA),short-hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), small nuclearRNA (snRNA or URNA), small nucleolar RNA (snoRNA), Small Cajal body RNA(scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), extracellular RNA(exRNA), repeat-derived RNA, transposable element RNA and longnon-coding RNA (lncRNA).

According to one embodiment, non-limiting examples of RNAi moleculesinclude, but are not limited to, small interfering RNA (siRNA), shorthairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA) andtrans-acting siRNA (tasiRNA).

As mentioned above, the methods of some embodiments of the invention areutilized to redirect a silencing activity and/or specificity of thenon-coding RNA molecule (or to generate a silencing activity and/orspecificity if the non-coding RNA molecule does not have an intrinsiccapability to silence a RNA molecule) towards a second target RNA ortowards a target RNA of interest.

According to one embodiment, the target RNA and the second target RNAare distinct.

According to one embodiment, the method of modifying a gene encoding orprocessed into a RNA silencing molecule to a target RNA in a eukaryoticcell, with the proviso that the eukaryotic cell is not a plant cell,comprises introducing into the eukaryotic cell a DNA editing agent whichredirects a silencing activity and/or specificity of the RNA silencingmolecule towards a second target RNA, the target RNA and the secondtarget RNA being distinct, thereby modifying the gene encoding the RNAsilencing molecule.

As used herein, the term “redirects a silencing specificity” refers toreprogramming the original specificity of the non-coding RNA (e.g. RNAsilencing molecule) towards a non-natural target of the non-coding RNA(e.g. RNA silencing molecule). Accordingly, the original specificity ofthe non-coding RNA is destroyed (i.e. loss of function) and the newspecificity is towards a RNA target distinct of the natural target (i.e.RNA of interest), i.e., gain of function. It will be appreciated thatonly gain of function occurs in cases that the non-coding RNA has nosilencing activity.

As used herein, the term “target RNA” refers to a RNA sequence naturallybound by a non-coding RNA molecule. Thus, the target RNA is consideredby the skilled artisan as a substrate for the non-coding RNA.

As used herein, the term “second target RNA” refers to a RNA sequence(coding or non-coding) not naturally bound by a non-coding RNA molecule.Thus, the second target RNA is not a natural substrate of the non-codingRNA.

As used herein, the term “target RNA of interest” refers to a RNAsequence (coding or non-coding) to be silenced by the designednon-coding RNA molecule.

As used herein, the phrase “silencing a target gene” refer to theabsence or observable reduction in the level of protein and/or mRNAproduct from the target gene. Thus, silencing of a target gene can be by5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% as comparedto a target gene not targeted by the designed non-coding RNA molecule ofthe invention.

The consequences of silencing can be confirmed by examination of theoutward properties of a eukaryotic cell or organism, or by biochemicaltechniques (as discussed below).

It will be appreciated that the designed non-coding RNA molecule of someembodiments of the invention can have some off-target specificityeffect/s provided that it does not affect the growth, differentiation orfunction of the eukaryotic cell or organism.

According to one embodiment, the second target RNA or target RNA ofinterest is endogenous to the eukaryotic cell. Exemplary endogenoussecond target RNA or target RNA of interest include, but are not limitedto, a product of a gene associated with cancer and/or apoptosis.Exemplary target genes associated with cancer include, but are notlimited to, p53, BAX, PUMA, NOXA and FAS genes as discussed in detailherein below.

According to one embodiment, the second target RNA or target RNA ofinterest is exogenous to the eukaryotic cell (also referred to herein asheterologous). In such a case, the second target RNA or target RNA ofinterest is a product of a gene that is not naturally part of theeukaryotic cell genome (i.e. which expresses the non-coding RNA).Exemplary exogenous target RNAs include, but are not limited to,products of a gene associated with an infectious disease, such as a geneof a pathogen (e.g. an insect, a virus, a bacteria, a fungi, anematode), as further discussed herein below. An exogenous target RNA(coding or non-coding) may comprise a nucleic acid sequence which sharessequence identity with an endogenous RNA sequence (e.g. may be partiallyhomologous to an endogenous nucleic acid sequence) of the eukaryoticorganism.

The specific binding of an endogenous non-coding RNA molecule with atarget RNA can be determined by computational algorithms (such as BLAST)and verified by methods including e.g. Northern blot, In Situhybridization, QuantiGene Plex Assay etc.

By use of the term “complementarity” or “complementary” is meant thatthe non-coding RNA molecule (or at least a portion of it that is presentin the processed small RNA form, or at least one strand of adouble-stranded polynucleotide or portion thereof, or a portion of asingle strand polynucleotide) hybridizes under physiological conditionsto the target RNA, or a fragment thereof, to effect regulation orfunction or suppression of the target gene. For example, in someembodiments, a non-coding RNA molecule has 100 percent sequence identityor at least about 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percentsequence identity when compared to a sequence of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 150, 200, 300, 400,500 or more contiguous nucleotides in the target RNA (or family membersof a given target gene).

As used herein, a non-coding RNA molecules, or their processed small RNAforms, are said to exhibit “complete complementarity” when everynucleotide of one of the sequences read 5′ to 3′ is complementary toevery nucleotide of the other sequence when read 3′ to 5′. A nucleotidesequence that is completely complementary to a reference nucleotidesequence will exhibit a sequence identical to the reverse complementsequence of the reference nucleotide sequence.

Methods for determining sequence complementarity are well known in theart and include, but not limited to, bioinformatics tools which are wellknown in the art (e.g. BLAST, multiple sequence alignment).

According to one embodiment, if the non-coding RNA molecule is orprocessed into a siRNA, the complementarity is in the range of 90-100%(e.g. 100%) to its target sequence.

According to one embodiment, if the non-coding RNA molecule is orprocessed into a miRNA or piRNA the complementarity is in the range of33-100% to its target sequence.

According to one embodiment, if the non-coding RNA molecule is a miRNA,the seed sequence complementarity (i.e. nucleotides 2-8 from the 5′) isin the range of 85-100% (e.g. 100%) to its target sequence.

According to one embodiment, the non-coding RNA can be further processedinto a small RNA form (e.g. pre-miRNA is processed into a mature miRNA).In such a case, homology is measured based on the processed small RNAform (e.g. the mature miRNA sequence).

As used herein, the term “small RNA form” refers to the mature small RNAbeing capable of hybridizing with a target RNA (or fragment thereof).According to one embodiment, the small RNA form has a silencingactivity.

According to one embodiment, the complementarity to the target sequenceis at least about 33% of the processed small RNA form (e.g. 33% of the21-24 nt). Thus, for example, if the non-coding RNA molecule is a miRNA,33% of the mature miRNA sequence (e.g. of the 21 nt) comprises seedcomplementation (e.g. 7 nt out of the 21 nt).

According to one embodiment, the complementarity to the target sequenceis at least about 45% of the processed small RNA form (e.g. 45% of the21-28 nt). Thus, for example, if the non-coding RNA molecule is a miRNA,45% of the mature miRNA sequence (e.g. 21 nt) comprises seedcomplementation (e.g. 9-10 nt out of the 21 nt).

According to one embodiment, the non-coding RNA (i.e. prior tomodification) is typically selected as one having about 10%, 20%, 30%,33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or up to 99%complementarity towards the sequence of the second target RNA or targetRNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than99% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than98% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than97% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than96% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than95% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than90% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than85% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than50% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (i.e.prior to modification) is typically selected as one having no more than33% complementarity towards the sequence of the second target RNA ortarget RNA of interest.

According to one embodiment, the non-coding RNA molecule (e.g. RNAsilencing molecule) is designed so as to comprise at least about 33%,40%, 45%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% complementarity towards the sequence of thesecond target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 33%complementarity towards the second target RNA or target RNA of interest(e.g. 85-100% seed match).

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 40%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 45%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 50%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 60%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 70%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 80%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 85%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 90%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 95%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 96%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 97%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 98%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise a minimum of 99%complementarity towards the second target RNA or target RNA of interest.

According to a specific embodiment, the non-coding RNA molecule (e.g.RNA silencing molecule) is designed so as to comprise 100%complementarity towards the second target RNA or target RNA of interest.

In order to generate silencing activity and/or specificity of anon-coding RNA molecule or redirect a silencing activity and/orspecificity of a non-coding RNA molecule (e.g. RNA silencing molecule)towards a second target RNA or target RNA of interest, the gene encodinga non-coding RNA molecule (e.g. RNA silencing molecule) is modifiedusing a DNA editing agent.

Following is a description of various non-limiting examples of methodsand DNA editing agents used to introduce nucleic acid alterations to agene encoding a non-coding RNA molecule (e.g. RNA silencing molecule)and agents for implementing same that can be used according to specificembodiments of the present disclosure.

Genome Editing using engineered endonucleases—this approach refers to areverse genetics method using artificially engineered nucleases totypically cut and create specific double-stranded breaks (DSBs) at adesired location(s) in the genome, which are then repaired by cellularendogenous processes such as, homologous recombination (HR) ornon-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends in adouble-stranded break (DSB) with or without minimal ends trimming, whileHR utilizes a homologous donor sequence as a template (i.e. the sisterchromatid formed during S-phase) for regenerating/copying the missingDNA sequence at the break site. In order to introduce specificnucleotide modifications to the genomic DNA, a donor DNA repair templatecontaining the desired sequence must be present during HR (exogenouslyprovided single stranded or double stranded DNA).

Genome editing cannot be performed using traditional restrictionendonucleases since most restriction enzymes recognize a few base pairson the DNA as their target and these sequences often will be found inmany locations across the genome resulting in multiple cuts which arenot limited to a desired location. To overcome this challenge and createsite-specific single- or double-stranded breaks (DSBs), several distinctclasses of nucleases have been discovered and bioengineered to date.These include the meganucleases, Zinc finger nucleases (ZFNs),transcription-activator like effector nucleases (TALENs) and CRISPR/Cas9(and all their variants) system.

Meganucleases—Meganucleases are commonly grouped into four families: theLAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNHfamily. These families are characterized by structural motifs, whichaffect catalytic activity and recognition sequence. For instance,members of the LAGLIDADG family are characterized by having either oneor two copies of the conserved LAGLIDADG motif. The four families ofmeganucleases are widely separated from one another with respect toconserved structural elements and, consequently, DNA recognitionsequence specificity and catalytic activity. Meganucleases are foundcommonly in microbial species and have the unique property of havingvery long recognition sequences (>14 bp) thus making them naturally veryspecific for cutting at a desired location.

This can be exploited to make site-specific double-stranded breaks(DSBs) in genome editing. One of skill in the art can use thesenaturally occurring meganucleases, however the number of such naturallyoccurring meganucleases is limited. To overcome this challenge,mutagenesis and high throughput screening methods have been used tocreate meganuclease variants that recognize unique sequences. Forexample, various meganucleases have been fused to create hybrid enzymesthat recognize a new sequence.

Alternatively, DNA interacting amino acids of the meganuclease can bealtered to design sequence specific meganucleases (see e.g., U.S. Pat.No. 8,021,867). Meganucleases can be designed using the methodsdescribed in e.g., Certo, M T et al. Nature Methods (2012) 9:073-975;U.S. Pat. Nos. 8,304,222; 8,021,867; 8,119,381; 8,124,369; 8,129,134;8,133,697; 8,143,015; 8,143,016; 8,148,098; or 8,163,514, the contentsof each are incorporated herein by reference in their entirety.Alternatively, meganucleases with site specific cutting characteristicscan be obtained using commercially available technologies e.g.,Precision Biosciences' Directed Nuclease Editor™ genome editingtechnology.

ZFNs and TALENs—Two distinct classes of engineered nucleases,zinc-finger nucleases (ZFNs) and transcription activator-like effectornucleases (TALENs), have both proven to be effective at producingtargeted double-stranded breaks (DSBs) (Christian et al., 2010; Kim etal., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

Basically, ZFNs and TALENs restriction endonuclease technology utilizesa non-specific DNA cutting enzyme which is linked to a specific DNAbinding domain (either a series of zinc finger domains or TALE repeats,respectively). Typically a restriction enzyme whose DNA recognition siteand cleaving site are separate from each other is selected. The cleavingportion is separated and then linked to a DNA binding domain, therebyyielding an endonuclease with very high specificity for a desiredsequence. An exemplary restriction enzyme with such properties is Fokl.Additionally Fokl has the advantage of requiring dimerization to havenuclease activity and this means the specificity increases dramaticallyas each nuclease partner recognizes a unique DNA sequence. To enhancethis effect, Fokl nucleases have been engineered that can only functionas heterodimers and have increased catalytic activity. The heterodimerfunctioning nucleases avoid the possibility of unwanted homodimeractivity and thus increase specificity of the double-stranded break(DSB).

Thus, for example to target a specific site, ZFNs and TALENs areconstructed as nuclease pairs, with each member of the pair designed tobind adjacent sequences at the targeted site. Upon transient expressionin cells, the nucleases bind to their target sites and the Fokl domainsheterodimerize to create a double-stranded break (DSB). Repair of thesedouble-stranded breaks (DSBs) through the non-homologous end-joining(NHEJ) pathway often results in small deletions or small sequenceinsertions (Indels). Since each repair made by NHEJ is unique, the useof a single nuclease pair can produce an allelic series with a range ofdifferent insertions or deletions at the target site.

In general NHEJ is relatively accurate (about 85% of DSBs in human cellsare repaired by NHEJ within about 30 min from detection) in gene editingerroneous NHEJ is relied upon as when the repair is accurate thenuclease will keep cutting until the repair product is mutagenic and therecognition/cut site/PAM motif is gone/mutated or that the transientlyintroduced nuclease is no longer present.

The deletions typically range anywhere from a few base pairs to a fewhundred base pairs in length, but larger deletions have beensuccessfully generated in cell culture by using two pairs of nucleasessimultaneously (Carlson et al., 2012; Lee et al., 2010). In addition,when a fragment of DNA with homology to the targeted region isintroduced in conjunction with the nuclease pair, the double-strandedbreak (DSB) can be repaired via homologous recombination (HR) togenerate specific modifications (Li et al., 2011; Miller et al., 2010;Urnov et al., 2005).

Although the nuclease portions of both ZFNs and TALENs have similarproperties, the difference between these engineered nucleases is intheir DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers andTALENs on TALEs. Both of these DNA recognizing peptide domains have thecharacteristic that they are naturally found in combinations in theirproteins. Cys2-His2 Zinc fingers are typically found in repeats that are3 bp apart and are found in diverse combinations in a variety of nucleicacid interacting proteins. TALEs on the other hand are found in repeatswith a one-to-one recognition ratio between the amino acids and therecognized nucleotide pairs. Because both zinc fingers and TALEs happenin repeated patterns, different combinations can be tried to create awide variety of sequence specificities. Approaches for makingsite-specific zinc finger endonucleases include, e.g., modular assembly(where Zinc fingers correlated with a triplet sequence are attached in arow to cover the required sequence), OPEN (low-stringency selection ofpeptide domains vs. triplet nucleotides followed by high-stringencyselections of peptide combination vs. the final target in bacterialsystems), and bacterial one-hybrid screening of zinc finger libraries,among others. ZFNs can also be designed and obtained commercially frome.g., Sangamo Biosciences™ (Richmond, Calif.).

Method for designing and obtaining TALENs are described in e.g. Reyon etal. Nature Biotechnology 2012 May; 30(5):460-5; Miller et al. NatBiotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research(2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2):149-53. A recently developed web-based program named Mojo Hand wasintroduced by Mayo Clinic for designing TAL and TALEN constructs forgenome editing applications (can be accessed throughwww(dot)talendesign(dot)org). TALEN can also be designed and obtainedcommercially from e.g., Sangamo Biosciences™ (Richmond, Calif.).

T-GEE system (TargetGene's Genome Editing Engine)—A programmablenucleoprotein molecular complex containing a polypeptide moiety and aspecificity conferring nucleic acid (SCNA) which assembles in-vivo, in atarget cell, and is capable of interacting with the predetermined targetnucleic acid sequence is provided. The programmable nucleoproteinmolecular complex is capable of specifically modifying and/or editing atarget site within the target nucleic acid sequence and/or modifying thefunction of the target nucleic acid sequence. Nucleoprotein compositioncomprises (a) polynucleotide molecule encoding a chimeric polypeptideand comprising (i) a functional domain capable of modifying the targetsite, and (ii) a linking domain that is capable of interacting with aspecificity conferring nucleic acid, and (b) specificity conferringnucleic acid (SCNA) comprising (i) a nucleotide sequence complementaryto a region of the target nucleic acid flanking the target site, and(ii) a recognition region capable of specifically attaching to thelinking domain of the polypeptide. The composition enables modifying apredetermined nucleic acid sequence target precisely, reliably andcost-effectively with high specificity and binding capabilities ofmolecular complex to the target nucleic acid through base-pairing ofspecificity-conferring nucleic acid and a target nucleic acid. Thecomposition is less genotoxic, modular in their assembly, utilize singleplatform without customization, practical for independent use outside ofspecialized core-facilities, and has shorter development time frame andreduced costs.

CRISPR-Cas system and all its variants (also referred to herein as“CRISPR”)—Many bacteria and archaea contain endogenous RNA-basedadaptive immune systems that can degrade nucleic acids of invadingphages and plasmids. These systems consist of clustered regularlyinterspaced short palindromic repeat (CRISPR) nucleotide sequences thatproduce RNA components and CRISPR associated (Cas) genes that encodeprotein components. The CRISPR RNAs (crRNAs) contain short stretches ofhomology to the DNA of specific viruses and plasmids and act as guidesto direct Cas nucleases to degrade the complementary nucleic acids ofthe corresponding pathogen. Studies of the type II CRISPR/Cas system ofStreptococcus pyogenes have shown that three components form aRNA/protein complex and together are sufficient for sequence-specificnuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairsof homology to the target sequence, and a trans-activating crRNA(tracrRNA) (Jinek et al. Science (2012) 337: 816-821).

It was further demonstrated that a synthetic chimeric guide RNA (gRNA)composed of a fusion between crRNA and tracrRNA could direct Cas9 tocleave DNA targets that are complementary to the crRNA in vitro. It wasalso demonstrated that transient expression of Cas9 in conjunction withsynthetic gRNAs can be used to produce targeted double-stranded breaks(DSBs) in a variety of different species (Cho et al., 2013; Cong et al.,2013; DiCarlo et al., 2013; Hwang et al., 2013a,b; Jinek et al., 2013;Mali et al., 2013).

The CRISPR/Cas system for genome editing contains two distinctcomponents: a gRNA and an endonuclease e.g. Cas9.

The gRNA (also referred to herein as short guide RNA (sgRNA)) istypically a 20-nucleotide sequence encoding a combination of the targethomologous sequence (crRNA) and the endogenous bacterial RNA that linksthe crRNA to the Cas9 nuclease (tracrRNA) in a single chimerictranscript. The gRNA/Cas9 complex is recruited to the target sequence bythe base-pairing between the gRNA sequence and the complement genomicDNA. For successful binding of Cas9, the genomic target sequence mustalso contain the correct Protospacer Adjacent Motif (PAM) sequenceimmediately following the target sequence. The binding of the gRNA/Cas9complex localizes the Cas9 to the genomic target sequence so that theCas9 can cut both strands of the DNA causing a double-strand break(DSB). Just as with ZFNs and TALENs, the double-stranded breaks (DSBs)produced by CRISPR/Cas can undergo homologous recombination or NHEJ andare susceptible to specific sequence modification during DNA repair.

The Cas9 nuclease has two functional domains: RuvC and HNH, each cuttinga different DNA strand. When both of these domains are active, the Cas9causes double strand breaks (DSBs) in the genomic DNA.

A significant advantage of CRISPR/Cas is that the high efficiency ofthis system is coupled with the ability to easily create syntheticgRNAs. This creates a system that can be readily modified to targetmodifications at different genomic sites and/or to target differentmodifications at the same site. Additionally, protocols have beenestablished which enable simultaneous targeting of multiple genes. Themajority of cells carrying the mutation present biallelic mutations inthe targeted genes.

However, apparent flexibility in the base-pairing interactions betweenthe gRNA sequence and the genomic DNA target sequence allows imperfectmatches to the target sequence to be cut by Cas9.

Modified versions of the Cas9 enzyme containing a single inactivecatalytic domain, either RuvC- or HNH-, are called ‘nickases’. With onlyone active nuclease domain, the Cas9 nickase cuts only one strand of thetarget DNA, creating a single-strand break or ‘nick’. A single-strandbreak, or nick, is mostly repaired by single strand break repairmechanism involving proteins such as but not only, PARP (sensor) andXRCC1/LIG III complex (ligation). If a single strand break (SSB) isgenerated by topoisomerase I poisons or by drugs that trap PARP1 onnaturally occurring SSBs then these could persist and when the cellenters into S-phase and the replication fork encounter such SSBs theywill become single ended DSBs which can only be repaired by HR. However,two proximal, opposite strand nicks introduced by a Cas9 nickase aretreated as a double-strand break, in what is often referred to as a‘double nick’ CRISPR system. A double-nick, which is basicallynon-parallel DSB, can be repaired like other DSBs by HR or NHEJdepending on the desired effect on the gene target and the presence of adonor sequence and the cell cycle stage (HR is of much lower abundanceand can only occur in S and G2 stages of the cell cycle). Thus, ifspecificity and reduced off-target effects are crucial, using the Cas9nickase to create a double-nick by designing two gRNAs with targetsequences in close proximity and on opposite strands of the genomic DNAwould decrease off-target effect as either gRNA alone will result innicks that are not likely to change the genomic DNA, even though theseevents are not impossible.

Modified versions of the Cas9 enzyme containing two inactive catalyticdomains (dead Cas9, or dCas9) have no nuclease activity while still ableto bind to DNA based on gRNA specificity. The dCas9 can be utilized as aplatform for DNA transcriptional regulators to activate or repress geneexpression by fusing the inactive enzyme to known regulatory domains.For example, the binding of dCas9 alone to a target sequence in genomicDNA can interfere with gene transcription.

There are a number of publicly available tools available to help chooseand/or design target sequences as well as lists of bioinformaticallydetermined unique gRNAs for different genes in different species, suchas but not limited to, the Feng Zhang lab's Target Finder, the MichaelBoutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, theCasFinder: Flexible algorithm for identifying specific Cas9 targets ingenomes and the CRISPR Optimal Target Finder.

In order to use the CRISPR system, both gRNA and a Cas endonuclease(e.g. Cas9) should be expressed or present (e.g., as a ribonucleoproteincomplex) in a target cell. The insertion vector can contain bothcassettes on a single plasmid or the cassettes are expressed from twoseparate plasmids. CRISPR plasmids are commercially available such asthe px330 plasmid from Addgene (75 Sidney St, Suite 550A•Cambridge,Mass. 02139). Use of clustered regularly interspaced short palindromicrepeats (CRISPR)-associated (Cas)-guide RNA technology and a Casendonuclease for modifying mammalian genomes are also at least disclosedby Bauer et al. [J Vis Exp. (2015) (95):e52118. doi: 10.3791/52118],which is specifically incorporated herein by reference in its entirety.Cas endonucleases that can be used to effect DNA editing with gRNAinclude, but are not limited to, Cas9, Cpf1 (Zetsche et al., 2015, Cell.163(3):759-71), C2c1, C2c2, and C2c3 (Shmakov et al., Mol Cell. 2015Nov. 5; 60(3):385-97).

According to a specific embodiment, the CRISPR comprises a short guideRNA (sgRNA) comprising a nucleic acid sequence as set forth in SEQ IDNOs: 5-6 or SEQ ID Nos 165-236.

“Hit and run” or “in-out”—involves a two-step recombination procedure.In the first step, an insertion-type vector containing a dualpositive/negative selectable marker cassette is used to introduce thedesired sequence alteration. The insertion vector contains a singlecontinuous region of homology to the targeted locus and is modified tocarry the mutation of interest. This targeting construct is linearizedwith a restriction enzyme at a one site within the region of homology,introduced into the cells, and positive selection is performed toisolate homologous recombination mediated events. The DNA carrying thehomologous sequence can be provided as a plasmid, single or doublestranded oligo. These homologous recombinants contain a localduplication that is separated by intervening vector sequence, includingthe selection cassette. In the second step, targeted clones aresubjected to negative selection to identify cells that have lost theselection cassette via intra-chromosomal recombination between theduplicated sequences. The local recombination event removes theduplication and, depending on the site of recombination, the alleleeither retains the introduced mutation or reverts to wild type. The endresult is the introduction of the desired modification without theretention of any exogenous sequences.

The “double-replacement” or “tag and exchange” strategy—involves atwo-step selection procedure similar to the hit and run approach, butrequires the use of two different targeting constructs. In the firststep, a standard targeting vector with 3′ and 5′ homology arms is usedto insert a dual positive/negative selectable cassette near the locationwhere the mutation is to be introduced. After the system components havebeen introduced to the cell and positive selection applied, HR mediatedevents could be identified. Next, a second targeting vector thatcontains a region of homology with the desired mutation is introducedinto targeted clones, and negative selection is applied to remove theselection cassette and introduce the mutation. The final allele containsthe desired mutation while eliminating unwanted exogenous sequences.

According to a specific embodiment, the DNA editing agent comprises aDNA targeting module (e.g., gRNA).

According to a specific embodiment, the DNA editing agent does notcomprise an endonuclease.

According to a specific embodiment, the DNA editing agent comprises anuclease (e.g. an endonuclease) and a DNA targeting module (e.g., gRNA).

According to a specific embodiment, the DNA editing agent is CRISPR/Cas,e.g. gRNA and Cas9.

According to a specific embodiment, the DNA editing agent is TALEN.

According to a specific embodiment, the DNA editing agent is ZFN.

According to a specific embodiment, the DNA editing agent ismeganuclease.

According to one embodiment, the DNA editing agent is linked to areporter for monitoring expression in a eukaryotic cell.

According to one embodiment, the reporter is a fluorescent reporterprotein.

The term “a fluorescent protein” refers to a polypeptide that emitsfluorescence and is typically detectable by flow cytometry, microscopyor any fluorescent imaging system, therefore can be used as a basis forselection of cells expressing such a protein.

Examples of fluorescent proteins that can be used as reporters are,without being limited to, the Green Fluorescent Protein (GFP), the BlueFluorescent Protein (BFP) and the red fluorescent proteins (e.g. dsRed,mCherry, RFP). A non-limiting list of fluorescent or other reportersincludes proteins detectable by luminescence (e.g. luciferase) orcolorimetric assay (e.g. GUS). According to a specific embodiment, thefluorescent reporter is a red fluorescent protein (e.g. dsRed, mCherry,RFP) or GFP.

A review of new classes of fluorescent proteins and applications can befound in Trends in Biochemical Sciences [Rodriguez, Erik A.; Campbell,Robert E.; Lin, John Y.; Lin, Michael Z.; Miyawaki, Atsushi; Palmer, AmyE.; Shu, Xiaokun; Zhang, Jin; Tsien, Roger Y. “The Growing and GlowingToolbox of Fluorescent and Photoactive Proteins”. Trends in BiochemicalSciences. doi: 10.1016/j.tibs. 2016.Sept.10].

According to another embodiment, the reporter is an antibiotic selectionmarker. Examples of antibiotic selection markers that can be used asreporters are, without being limited to, neomycin phosphotransferase II(nptII) and hygromycin phosphotransferase (hpt). Additional marker geneswhich can be used in accordance with the present teachings include, butare not limited to, gentamycin acetyltransferase (accC3) resistance andbleomycin and phleomycin resistance genes.

It will be appreciated that the enzyme NPTII inactivates byphosphorylation a number of aminoglycoside antibiotics such askanamycin, neomycin, geneticin (or G418) and paromomycin. Of these, G418is routinely used for selection of transformed mammalian cells.

Regardless of the DNA editing agent used, the method of the invention isemployed such that the gene encoding the non-coding RNA molecule (e.g.RNA silencing molecule) is modified by at least one of a deletion, aninsertion or a point mutation.

According to one embodiment, the modification is in a structured regionof the non-coding RNA molecule or the RNA silencing molecule.

According to one embodiment, the modification is in a stem region of thenon-coding RNA molecule or the RNA silencing molecule.

According to one embodiment, the modification is in a loop region of thenon-coding RNA molecule or the RNA silencing molecule.

According to one embodiment, the modification is in a stem region and aloop region of the non-coding RNA molecule or the RNA silencingmolecule.

According to one embodiment, the modification is in a non-structuredregion of the non-coding RNA molecule or the RNA silencing molecule.

According to one embodiment, the modification is in a stem region and aloop region and in non-structured region of the non-coding RNA moleculeor the RNA silencing molecule.

According to a specific embodiment, the modification comprises amodification of about 10-250 nucleotides, about 10-200 nucleotides,about 10-150 nucleotides, about 10-100 nucleotides, about 10-50nucleotides, about 1-50 nucleotides, about 1-10 nucleotides, about50-150 nucleotides, about 50-100 nucleotides or about 100-200nucleotides (as compared to the native non-coding RNA molecule, e.g. RNAsilencing molecule).

According to one embodiment, the modification comprises a modificationof at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 orat most 250 nucleotides (as compared to the native non-coding RNAmolecule, e.g. RNA silencing molecule).

According to one embodiment, the modification can be in a consecutivenucleic acid sequence (e.g. at least 5, 10, 20, 30, 40, 50, 100, 150,200 bases).

According to one embodiment, the modification can be in anon-consecutive manner, e.g. throughout a 20, 50, 100, 150, 200, 500,1000 nucleic acid sequence.

According to a specific embodiment, the modification comprises amodification of at most 200 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 150 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 100 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 50 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 25 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 20 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 15 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 10 nucleotides.

According to a specific embodiment, the modification comprises amodification of at most 5 nucleotides.

According to one embodiment, the modification depends on the structureof the RNA silencing molecule.

Accordingly, when the RNA silencing molecule contains a non-essentialstructure (i.e. a secondary structure of the RNA silencing moleculewhich does not play a role in its proper biogenesis and/or function) oris purely dsRNA (i.e. the RNA silencing molecule having a perfect oralmost perfect dsRNA), a few modifications (e.g. 20-30 nucleotides, e.g.1-10 nucleotides, e.g. 5 nucleotides) are introduced in order toredirect the silence specificity of the RNA silencing molecule.

According to another embodiment, when the RNA silencing molecule has anessential structure (i.e. the proper biogenesis and/or activity of theRNA silencing molecule is dependent on its secondary structure), largermodifications (e.g. 10-200 nucleotides, e.g. 50-150 nucleotides, e.g.,more than 30 nucleotides and not exceeding 200 nucleotides, 30-200nucleotides, 35-200 nucleotides, 35-150 nucleotides, 35-100 nucleotides)are introduced in order to redirect the silence specificity of the RNAsilencing molecule.

According to one embodiment, the modification is such that therecognition/cut site/PAM motif of the RNA silencing molecule is modifiedto abolish the original PAM recognition site.

According to a specific embodiment, the modification is in at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids in a PAM motif.

According to one embodiment, the modification comprises an insertion.

According to a specific embodiment, the insertion comprises an insertionof about 10-250 nucleotides, about 10-200 nucleotides, about 10-150nucleotides, about 10-100 nucleotides, about 10-50 nucleotides, about1-50 nucleotides, about 50-150 nucleotides, about 50-100 nucleotides orabout 100-200 nucleotides (as compared to the native non-coding RNAmolecule, e.g. RNA silencing molecule).

According to a specific embodiment, the insertion comprises an insertionof about 10-250 nucleotides, about 10-200 nucleotides, about 10-150nucleotides, about 10-100 nucleotides, about 10-50 nucleotides, about1-50 nucleotides, about 1-10 nucleotides, about 50-150 nucleotides,about 50-100 nucleotides or about 100-200 nucleotides (as compared tothe native non-coding RNA molecule, e.g. RNA silencing molecule).

According to one embodiment, the insertion comprises an insertion of atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or at most250 nucleotides (as compared to the native non-coding RNA molecule, e.g.RNA silencing molecule).

According to a specific embodiment, the insertion comprises an insertionof at most 200 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 150 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 100 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 50 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 25 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 20 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 15 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 10 nucleotides.

According to a specific embodiment, the insertion comprises an insertionof at most 5 nucleotides.

According to one embodiment, the modification comprises a deletion.

According to a specific embodiment, the deletion comprises a deletion ofabout 10-250 nucleotides, about 10-200 nucleotides, about 10-150nucleotides, about 10-100 nucleotides, about 10-50 nucleotides, about1-50 nucleotides, about 1-10 nucleotides, about 50-150 nucleotides,about 50-100 nucleotides or about 100-200 nucleotides (as compared tothe native non-coding RNA molecule, e.g. RNA silencing molecule).

According to one embodiment, the deletion comprises a deletion of atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or at most250 nucleotides (as compared to the native non-coding RNA molecule, e.g.RNA silencing molecule).

According to a specific embodiment, the deletion comprises a deletion ofat most 200 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 150 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 100 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 50 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 25 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 20 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 15 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 10 nucleotides.

According to a specific embodiment, the deletion comprises a deletion ofat most 5 nucleotides.

According to one embodiment, the modification comprises a pointmutation.

According to a specific embodiment, the point mutation comprises a pointmutation of about 10-250 nucleotides, about 10-200 nucleotides, about10-150 nucleotides, about 10-100 nucleotides, about 10-50 nucleotides,about 1-50 nucleotides, about 1-10 nucleotides, about 50-150nucleotides, about 50-100 nucleotides or about 100-200 nucleotides (ascompared to the native non-coding RNA molecule, e.g. RNA silencingmolecule).

According to one embodiment, the point mutation comprises a pointmutation in at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 or at most 250 nucleotides (as compared to the nativenon-coding RNA molecule, e.g. RNA silencing molecule).

According to a specific embodiment, the point mutation comprises a pointmutation in at most 200 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 150 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 100 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 50 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 25 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 20 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 15 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 10 nucleotides.

According to a specific embodiment, the point mutation comprises a pointmutation in at most 5 nucleotides.

According to one embodiment, the modification comprises a combination ofany of a deletion, an insertion and/or a point mutation.

According to one embodiment, the modification comprises nucleotidereplacement (e.g. nucleotide swapping).

According to a specific embodiment, the swapping comprises swapping ofabout 10-250 nucleotides, about 10-200 nucleotides, about 10-150nucleotides, about 10-100 nucleotides, about 10-50 nucleotides, about1-50 nucleotides, about 1-10 nucleotides, about 50-150 nucleotides,about 50-100 nucleotides or about 100-200 nucleotides (as compared tothe native non-coding RNA molecule, e.g. RNA silencing molecule).

According to one embodiment, the nucleotide swapping comprises anucleotide replacement in at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200 or at most 250 nucleotides (as compared to the nativenon-coding RNA molecule, e.g. RNA silencing molecule).

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 200 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 150 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 100 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 50 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 25 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 20 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 15 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 10 nucleotides.

According to a specific embodiment, the nucleotide swapping comprises anucleotide replacement in at most 5 nucleotides.

According to one embodiment, the gene encoding the non-coding RNAmolecule (e.g. RNA silencing molecule) is modified by swapping asequence of an endogenous RNA silencing molecule (e.g. miRNA) with a RNAsilencing sequence of choice (e.g. siRNA).

According to a specific embodiment, the sequence of a siRNA used forgene swapping of an endogenous RNA silencing molecule (e.g. miRNA)comprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1-4, SEQ ID Nos: 93-164 or SEQ ID Nos 243-252. According toone embodiment, the guide strand of the non-coding RNA molecule (e.g.RNA silencing molecule) is modified to preserve originality of structureand keep the same base pairing profile.

According to one embodiment, the passenger strand of the non-coding RNAmolecule (e.g. RNA silencing molecule) is modified to preserveoriginality of structure and keep the same base pairing profile.

As used herein, the term “originality of structure” refers to thesecondary RNA structure (i.e. base pairing profile). Keeping theoriginality of structure is important for correct and efficientbiogenesis/processing of the non-coding RNA (e.g. RNA silencing moleculesuch as siRNA or miRNA) that is structure- and not purelysequence-dependent.

According to one embodiment, the non-coding RNA (e.g. RNA silencingmolecule) is modified in the guide strand (silencing strand) as tocomprise about 50-100% complementarity to the target RNA (as discussedabove) while the passenger strand is modified to preserve the original(unmodified) non-coding RNA structure.

According to one embodiment, the non-coding RNA (e.g. RNA silencingmolecule) is modified such that the seed sequence (e.g. for miRNAnucleotides 2-8 from the 5′ terminal) is complimentary to the targetsequence.

According to a specific embodiment, the RNA silencing molecule (i.e.RNAi molecule) is designed such that a sequence of the RNAi molecule ismodified to preserve originality of structure and to be recognized bycellular RNAi processing and executing factors.

The DNA editing agent of the invention may be introduced into eukaryoticcells using DNA delivery methods (e.g. by expression vectors) or usingDNA-free methods.

According to one embodiment, the gRNA (or any other DNA recognitionmodule used, dependent on the DNA editing system that is used) can beprovided as RNA to the cell.

Thus, it will be appreciated that the present techniques relate tointroducing the DNA editing agent using DNA-free methods such as RNAtransfection (e.g. mRNA+gRNA transfection), or Ribonucleoprotein (RNP)transfection (e.g. protein-RNA complex transfection, e.g. Cas9/gRNA RNPcomplex transfection, or any combination of DNA/RNA/Proteins).

For example, Cas9 can be introduced as a DNA expression plasmid, invitro transcript (i.e. RNA), or as a recombinant protein bound to theRNA portion in a ribonucleoprotein particle (RNP). gRNA, for example,can be delivered either as a DNA plasmid or as an in vitro transcript(i.e. RNA).

Any method known in the art for RNA or RNP transfection can be used inaccordance with the present teachings, such as, but not limited tomicroinjection [as described by Cho et al., “Heritable gene knockout inCaenorhabditis elegans by direct injection of Cas9-sgRNAribonucleoproteins,” Genetics (2013) 195:1177-1180, incorporated hereinby reference], electroporation [as described by Kim et al., “Highlyefficient RNA-guided genome editing in human cells via delivery ofpurified Cas9 ribonucleoproteins” Genome Res. (2014) 24:1012-1019,incorporated herein by reference], or lipid-mediated transfection e.g.using liposomes [as described by Zuris et al., “Cationic lipid-mediateddelivery of proteins enables efficient protein-based genome editing invitro and in vivo” Nat Biotechnol. (2014) doi: 10.1038/nbt.3081,incorporated herein by reference]. Additional methods of RNAtransfection are described in U.S. Patent Application No. 20160289675,incorporated herein by reference in its entirety.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and vector-free. A RNA transgenecan be delivered to a cell and expressed therein, as a minimalexpressing cassette without the need for any additional sequences (e.g.viral sequences).

According to one embodiment, for expression of exogenous DNA editingagents of the invention in mammalian cells, a polynucleotide sequenceencoding the DNA editing agent is ligated into a nucleic acid constructsuitable for mammalian cell expression. Such a nucleic acid constructincludes a promoter sequence for directing transcription of thepolynucleotide sequence in the cell in a constitutive or induciblemanner.

The nucleic acid construct (also referred to herein as an “expressionvector”) of some embodiments of the invention includes additionalsequences which render this vector suitable for replication andintegration in eukaryotes (e.g., shuttle vectors). In addition, typicalcloning vectors may also contain a transcription and translationinitiation sequence, transcription and translation terminator and apolyadenylation signal. By way of example, such constructs willtypically include a 5′ LTR, a tRNA binding site, a packaging signal, anorigin of second-strand DNA synthesis, and a 3′ LTR or a portionthereof.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of someembodiments of the invention is active in the specific cell populationtransformed. Examples of cell type-specific and/or tissue-specificpromoters include promoters such as albumin that is liver specific[Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specificpromoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; inparticular promoters of T-cell receptors [Winoto et al., (1989) EMBO J.8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740],neuron-specific promoters such as the neurofilament promoter [Byrne etal. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specificpromoters [Edlunch et al. (1985) Science 230:912-916] or mammarygland-specific promoters such as the milk whey promoter (U.S. Pat. No.4,873,316 and European Application Publication No. 264,166).

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for some embodiments of the inventioninclude those derived from polyoma virus, human or murinecytomegalovirus (CMV), the long term repeat from various retrovirusessuch as murine leukemia virus, murine or Rous sarcoma virus and HIV.See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of mRNA translation. Two distinctsequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for some embodiments of theinvention include those derived from SV40.

In addition to the elements already described, the expression vector ofsome embodiments of the invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote the extra chromosomal replication ofthe viral genome in permissive cell types. Plasmids bearing these viralreplicons are replicated episomally as long as the appropriate factorsare provided by genes either carried on the plasmid or with the genomeof the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of some embodiments of the invention can furtherinclude additional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

It will be appreciated that the individual elements comprised in theexpression vector can be arranged in a variety of configurations. Forexample, enhancer elements, promoters and the like, and even thepolynucleotide sequence(s) encoding a DNA editing agent can be arrangedin a “head-to-tail” configuration, may be present as an invertedcomplement, or in a complementary configuration, as an anti-parallelstrand. While such variety of configuration is more likely to occur withnon-coding elements of the expression vector, alternative configurationsof the coding sequence within the expression vector are also envisioned.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Stratagene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMT010/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Viruses are very specialized infectious agents that have evolved, inmany cases, to elude host defense mechanisms. Typically, viruses infectand propagate in specific cell types. The targeting specificity of viralvectors utilizes its natural specificity to specifically targetpredetermined cell types and thereby introduce a recombinant gene intothe infected cell. Thus, the type of vector used by some embodiments ofthe invention will depend on the cell type transformed. The ability toselect suitable vectors according to the cell type transformed is wellwithin the capabilities of the ordinary skilled artisan and as such nogeneral description of selection consideration is provided herein. Forexample, bone marrow cells can be targeted using the human T cellleukemia virus type I (HTLV-I) and kidney cells may be targeted usingthe heterologous promoter present in the baculovirus Autographacalifornica nucleopolyhedrovirus (AcMNPV) as described in Liang C Y etal., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression of DNAediting agents since they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the life cycleof, for example, retrovirus and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. This contrasts with vertical-type of infection in which theinfectious agent spreads only through daughter progeny. Viral vectorscan also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

According to one embodiment, in order to express a functional DNAediting agent, in cases where the cleaving module (nuclease) is not anintegral part of the DNA recognition unit, the expression vector mayencode the cleaving module as well as the DNA recognition unit (e.g.gRNA in the case of CRISPR/Cas).

Alternatively, the cleaving module (nuclease) and the DNA recognitionunit (e.g. gRNA) may be cloned into separate expression vectors. In sucha case, at least two different expression vectors must be transformedinto the same eukaryotic cell.

Alternatively, when a nuclease is not utilized (i.e. not administeredfrom an exogenous source to the cell), the DNA recognition unit (e.g.gRNA) may be cloned and expressed using a single expression vector.

According to one embodiment, the DNA editing agent comprises a nucleicacid agent encoding at least one DNA recognition unit (e.g. gRNA)operatively linked to a cis-acting regulatory element active ineukaryotic cells (e.g., promoter).

According to one embodiment, the nuclease (e.g. endonuclease) and theDNA recognition unit (e.g. gRNA) are encoded from the same expressionvector. Such a vector may comprise a single cis-acting regulatoryelement active in eukaryotic cells (e.g., promoter) for expression ofboth the nuclease and the DNA recognition unit. Alternatively, thenuclease and the DNA recognition unit may each be operably linked to acis-acting regulatory element active in eukaryotic cells (e.g.,promoter).

According to one embodiment, the nuclease (e.g. endonuclease) and theDNA recognition unit (e.g. gRNA) are encoded from different expressionvectors whereby each is operably linked to a cis-acting regulatoryelement active in eukaryotic cells (e.g., promoter).

According to one embodiment, the method of some embodiments of theinvention further comprises introducing into the eukaryotic cell donoroligonucleotides.

According to one embodiment, when the modification is an insertion, themethod further comprises introducing into the eukaryotic cell donoroligonucleotides.

According to one embodiment, when the modification is a deletion, themethod further comprises introducing into the eukaryotic cell donoroligonucleotides.

According to one embodiment, when the modification is a deletion andinsertion (e.g. swapping), the method further comprises introducing intothe eukaryotic cell donor oligonucleotides.

According to one embodiment, when the modification is a point mutation,the method further comprises introducing into the eukaryotic cell donoroligonucleotides.

As used herein, the term “donor oligonucleotides” or “donor oligos”refers to exogenous nucleotides, i.e. externally introduced into theeukaryotic cell to generate a precise change in the genome. According toone embodiment, the donor oligonucleotides are synthetic.

According to one embodiment, the donor oligos are RNA oligos.

According to one embodiment, the donor oligos are DNA oligos.

According to one embodiment, the donor oligos are synthetic oligos.

According to one embodiment, the donor oligonucleotides comprisesingle-stranded donor oligonucleotides (ssODN).

According to one embodiment, the donor oligonucleotides comprisedouble-stranded donor oligonucleotides (dsODN).

According to one embodiment, the donor oligonucleotides comprisedouble-stranded DNA (dsDNA).

According to one embodiment, the donor oligonucleotides comprisedouble-stranded DNA-RNA duplex (DNA-RNA duplex).

According to one embodiment, the donor oligonucleotides comprisedouble-stranded DNA-RNA hybrid

According to one embodiment, the donor oligonucleotides comprisesingle-stranded DNA-RNA hybrid

According to one embodiment, the donor oligonucleotides comprisesingle-stranded DNA (ssDNA).

According to one embodiment, the donor oligonucleotides comprisedouble-stranded RNA (dsRNA).

According to one embodiment, the donor oligonucleotides comprisesingle-stranded RNA (s sRNA).

According to one embodiment, the donor oligonucleotides comprise the DNAor RNA sequence for swapping (as discussed above).

According to one embodiment, the donor oligonucleotides are provided ina non-expressed vector format or oligo.

According to one embodiment, the donor oligonucleotides comprise a DNAdonor plasmid.

According to one embodiment, the donor oligonucleotides comprise about50-5000, about 100-5000, about 250-5000, about 500-5000, about 750-5000,about 1000-5000, about 1500-5000, about 2000-5000, about 2500-5000,about 3000-5000, about 4000-5000, about 50-4000, about 100-4000, about250-4000, about 500-4000, about 750-4000, about 1000-4000, about1500-4000, about 2000-4000, about 2500-4000, about 3000-4000, about50-3000, about 100-3000, about 250-3000, about 500-3000, about 750-3000,about 1000-3000, about 1500-3000, about 2000-3000, about 50-2000, about100-2000, about 250-2000, about 500-2000, about 750-2000, about1000-2000, about 1500-2000, about 50-1000, about 100-1000, about250-1000, about 500-1000, about 750-1000, about 50-750, about 150-750,about 250-750, about 500-750, about 50-500, about 150-500, about200-500, about 250-500, about 350-500, about 50-250, about 150-250, orabout 200-250 nucleotides.

According to a specific embodiment, the donor oligonucleotidescomprising the ssODN (e.g. ssDNA or ssRNA) comprise about 200-500nucleotides.

According to a specific embodiment, the donor oligonucleotidescomprising the dsODN (e.g. dsDNA or dsRNA) comprise about 250-5000nucleotides.

According to one embodiment, for gene swapping of an endogenous RNAsilencing molecule (e.g. miRNA) with a RNA silencing sequence of choice(e.g. siRNA), the expression vector, ssODN (e.g. ssDNA or ssRNA) ordsODN (e.g. dsDNA or dsRNA) need not to be expressed in a eukaryoticcell and only serves as a non-expressing template. According to aspecific embodiment, in such a case only the DNA editing agent (e.g.Cas9/sgRNA modules) need to be expressed if provided in a DNA form.

According to some embodiments, for gene editing of an endogenousnon-coding RNA molecule (e.g. RNA silencing molecule) without the use ofa nuclease, the DNA editing agent (e.g., gRNA) may be introduced intothe eukaryotic cell with or without donor oligonucleotides (as discussedherein).

According to one embodiment, introducing into the eukaryotic cell donoroligonucleotides is effected using any of the methods described above(e.g. using the expression vectors or RNP transfection).

According to one embodiment, the gRNA and the DNA donor oligonucleotidesare co-introduced into the eukaryotic cell. It will be appreciated thatany additional factors (e.g. nuclease) may be co-introduced therewith.

According to one embodiment, the gRNA is introduced into the eukaryoticcell prior to the DNA donor oligonucleotides (e.g. within a few minutesor a few hours). It will be appreciated that any additional factors(e.g. nuclease) may be introduced prior to, concomitantly with, orfollowing the gRNA or the DNA donor oligonucleotides.

According to one embodiment, the gRNA is introduced into the eukaryoticcell subsequent to the DNA donor oligonucleotides (e.g. within a fewminutes or a few hours). It will be appreciated that any additionalfactors (e.g. nuclease) may be introduced prior to, concomitantly with,or following the gRNA or the DNA donor oligonucleotides.

According to one embodiment, there is provided a composition comprisingat least one gRNA and DNA donor oligonucleotides for genome editing.

According to one embodiment, there is provided a composition comprisingat least one gRNA, a nuclease (e.g. endonuclease) and DNA donoroligonucleotides for genome editing.

Various methods can be used to introduce the expression vector or donoroligos of some embodiments of the invention into eukaryotic cells (e.g.stem cells). Such methods are generally described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,New York (1989, 1992), in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al.,Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al.,Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, Butterworths, Boston Mass.(1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. For gene therapy, thepreferred constructs are viruses, most preferably adenoviruses, AAV,lentiviruses, or retroviruses. A viral construct such as a retroviralconstruct includes at least one transcriptional promoter/enhancer orlocus-defining element(s), or other elements that control geneexpression by other means such as alternate splicing, nuclear RNAexport, or post-translational modification of messenger. Such vectorconstructs also include a packaging signal, long terminal repeats (LTRs)or portions thereof, and positive and negative strand primer bindingsites appropriate to the virus used, unless it is already present in theviral construct. In addition, such a construct typically includes asignal sequence for secretion of the peptide from a host cell in whichit is placed. Preferably the signal sequence for this purpose is amammalian signal sequence or the signal sequence of the polypeptidevariants of some embodiments of the invention. Optionally, the constructmay also include a signal that directs polyadenylation, as well as oneor more restriction sites and a translation termination sequence. By wayof example, such constructs will typically include a 5′ LTR, a tRNAbinding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof. Other vectors can be usedthat are non-viral, such as cationic lipids, polylysine, and dendrimers.

Other than containing the necessary elements for the transcription andtranslation of the inserted coding sequence, the expression construct ofsome embodiments of the invention can also include sequences engineeredto enhance stability, production, purification, yield or toxicity of theexpressed peptide.

According to a specific embodiment, a bombardment method is used tointroduce foreign genes into eukaryotic cells. According to oneembodiment, the method is transient. An exemplary bombardment methodwhich can be used in accordance with some embodiments of the inventionis discussed in the examples section which follows. Bombardment ofeukaryotic cells (e.g. mammalian cells) is also taught by Uchida M etal., Biochim Biophys Acta. (2009) 1790(8):754-64, incorporated herein byreference.

Regardless of the transformation/infection method employed, the presentteachings further select transformed cells comprising a genome editingevent.

According to a specific embodiment, selection is carried out such thatonly cells comprising a successful accurate modification (e.g. swapping,insertion, deletion, point mutation) in the specific locus are selected.Accordingly, cells comprising any event that includes a modification(e.g. an insertion, deletion, point mutation) in an unintended locus arenot selected.

According to one embodiment, selection of modified cells can beperformed at the phenotypic level, by detection of a molecular event, bydetection of a fluorescent reporter, or by growth in the presence ofselection (e.g., antibiotic or other selection marker such as resistanceto a drug i.e. Nutlin3 in the case of TP53 silencing).

According to one embodiment, selection of modified cells is performed byanalyzing the biogenesis and occurrence of the newly edited non-codingRNA molecule (e.g. the presence of new miRNA version, the presence ofnovel edited siRNAs, piRNAs, tasiRNAs, etc).

According to one embodiment, selection of modified cells is performed byanalyzing the silencing activity and/or specificity of the non-codingRNA molecule (e.g. RNA silencing molecule) towards a second target RNAor target RNA of interest by validating at least one eukaryotic cell ororganism phenotype of the organism that encode the target RNA e.g. cellsize, growth rate/inhibition, cell shape, cell membrane integrity, tumorsize, tumor shape, a pigmentation of an organism, infection parametersin an organism (such as viral load or bacterial load) or inflammationparameters in an organism (such as fever or redness).

According to one embodiment, the silencing specificity of the non-codingRNA molecule is determined genotypically, e.g. by expression of a geneor lack of expression.

According to one embodiment, the silencing specificity of the non-codingRNA molecule is determined phenotypically.

According to one embodiment, a phenotype of the eukaryotic cell ororganism is determined prior to a genotype.

According to one embodiment, a genotype of the eukaryotic cell ororganism is determined prior to a phenotype.

According to one embodiment, selection of modified cells is performed byanalyzing the silencing activity and/or specificity of the non-codingRNA molecule (e.g. RNA silencing molecule) towards a second target RNAor target RNA of interest by measuring a RNA level of the second targetRNA or target RNA of interest. This can be effected using any methodknown in the art, e.g. by Northern blotting, Nuclease Protection Assays,In Situ hybridization, quantitative RT-PCR or immunoblotting.

According to one embodiment, selection of modified cells is performed byanalyzing eukaryotic cells or clones comprising the DNA editing eventalso referred to herein as “mutation” or “edit”, dependent on the typeof editing sought e.g., insertion, deletion, insertion-deletion (Indel),inversion, substitution and combinations thereof.

Methods for detecting sequence alteration are well known in the art andinclude, but not limited to, DNA and RNA sequencing (e.g., nextgeneration sequencing), electrophoresis, an enzyme-based mismatchdetection assay and a hybridization assay such as PCR, RT-PCR, RNaseprotection, in-situ hybridization, primer extension, Southern blot,Northern Blot and dot blot analysis. Various methods used for detectionof single nucleotide polymorphisms (SNPs) can also be used, such as PCRbased T7 endonuclease, Hetroduplex and Sanger sequencing, or PCRfollowed by restriction digest to detect appearance or disappearance ofunique restriction site/s.

Another method of validating the presence of a DNA editing event e.g.,Indels comprises a mismatch cleavage assay that makes use of a structureselective enzyme (e.g. endonuclease) that recognizes and cleavesmismatched DNA.

According to one embodiment, selection of transformed cells is effectedby flow cytometry (FACS) selecting transformed cells exhibitingfluorescence emitted by the fluorescent reporter. Following FACSsorting, positively selected pools of transformed eukaryotic cells,displaying the fluorescent marker are collected and an aliquot can beused for testing the DNA editing event as discussed above.

In cases where antibiotic selection marker was used, followingtransformation eukaryotic cell are cultivated in the presence ofselection (e.g., antibiotic), e.g. in a cell culture. A portion of thecells of the cell culture are then analyzed (validated) for the DNAediting event, as discussed above.

According to one embodiment of the invention, the method furthercomprises validating in the transformed cells complementarity of theendogenous non-coding RNA molecule (e.g. RNA silencing molecule) towardsthe second target RNA.

As mentioned above, following modification of the gene encoding thenon-coding RNA molecule (e.g. RNA silencing molecule), the non-codingRNA molecule (e.g. RNA silencing molecule) comprises at least about 30%,33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% complementarity towards the sequence of thesecond target RNA or target RNA of interest.

The specific binding of designed non-coding RNA molecule with a targetRNA of interest can be determined by any method known in the art, suchas by computational algorithms (e.g. BLAST) and verified by methodsincluding e.g. Northern blot, In Situ hybridization, QuantiGene PlexAssay etc.

It will be appreciated that positive eukaryotic cells can be homozygousor heterozygous for the DNA editing event. In case of a heterozygouscell, the cell may comprise a copy of a modified gene and a copy of anon-modified gene of the non-coding RNA molecule (e.g. RNA silencingmolecule). The skilled artisan will select the cells for furtherculturing/regeneration according to the intended use.

According to one embodiment, when a transient method is desired,eukaryotic cells exhibiting the presence of a DNA editing event asdesired are further analyzed and selected for the presence of the DNAediting agent, namely, loss of DNA sequences encoding for the DNAediting agent. This can be done, for example, by analyzing the loss ofexpression of the DNA editing agent (e.g., at the mRNA, protein) e.g.,by fluorescent detection of GFP or q-PCR, HPLC.

According to one embodiment, when a transient method is desired, theeukaryotic cells may be analyzed for the presence of the nucleic acidconstruct as described herein or portions thereof e.g., nucleic acidsequence encoding the DNA editing agent. This can be affirmed byfluorescent microscopy, q-PCR, FACS, and or any other method such asSouthern blot, PCR, sequencing, HPLC).

Positive eukaryotic cell clones may be stored (e.g., cryopreserved).

Alternatively, eukaryotic cells may be further cultured and maintained,for example, in an undifferentiated state for extended periods of timeor may be induced to differentiate into other cell types, tissues,organs or organisms as required.

The DNA editing agents and optionally the donor oligos of someembodiments of the invention can be administered to a single cell, to agroup of cells (e.g. primary cells or cell lines as discussed above) orto an organism (e.g. mammal, bird, fish, and insect, as discussedabove).

Accordingly, the DNA editing agents and optionally the donor oligos ofsome embodiments of the invention (or expression vectors or RNP complexcomprising same) can be administered to an organism per se, or in apharmaceutical composition where it is mixed with suitable carriers orexcipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the DNA editing agents andoptionally the donor oligos accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of some embodiments of the invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodimentsof the invention thus may be formulated in conventional manner using oneor more physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinylpyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to some embodiments of the invention are convenientlydelivered in the form of an aerosol spray presentation from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of some embodiments of the invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of someembodiments of the invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, a therapeutically effective amount means anamount of active ingredients (DNA editing agent) effective to prevent,alleviate or ameliorate symptoms of a disorder (e.g., cancer orinfectious disease) or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Animal models for cancerous diseases are described e.g. in Yee et al.,Cancer Growth Metastasis. (2015) 8(Suppl 1): 115-118. Animal models forinfectious diseases are described e.g. in Shevach, Current Protocols inImmunology, Published Online: 1 Apr. 2011, DOI:10.1002/0471142735.im1900s93.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provide theactive ingredient at a sufficient amount to induce or suppress thebiological effect (minimal effective concentration, MEC). The MEC willvary for each preparation, but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

The DNA editing agent designed to comprise a silencing specificity of anon-coding RNA molecule towards a target RNA of interest can be used fortreating various diseases and conditions as discussed below.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

As used herein, the term “subject” or “subject in need thereof” includesanimals, including mammals, preferably human beings, at any age orgender which suffer from the pathology. Preferably, this termencompasses individuals who are at risk to develop the pathology.

According to one aspect of the invention, there is provided a method oftreating an infectious disease in a subject in need thereof, the methodcomprising modifying a gene encoding or processed into a non-coding RNAmolecule or encoding or processed into an RNA silencing moleculeaccording to the method of some embodiments of the invention, whereinthe target RNA of interest is associated with onset or progression ofthe infectious disease, thereby treating the infectious disease in thesubject.

According to one aspect of the invention, there is provided a DNAediting agent conferring a silencing specificity of a non-coding RNAmolecule having no RNA silencing activity towards a target RNA ofinterest, wherein the target RNA of interest is associated with onset orprogression of an infectious disease, for use in treating an infectiousdisease in a subject in need thereof.

According to one aspect of the invention, there is provided a DNAediting agent redirecting a silencing specificity of a gene encoding orprocessed into a RNA silencing molecule to a target RNA towards a secondtarget RNA, the target RNA and the second target RNA being distinct,wherein the second target RNA is associated with onset or progression ofan infectious disease, for use in treating an infectious disease in asubject in need thereof.

The term “infectious diseases” as used herein refers to any of chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

According to one embodiment, in order to treat an infectious disease ina subject, the non-coding RNA molecule (e.g. RNA silencing molecule) isdesigned to target a RNA of interest associated with onset orprogression of the infectious disease.

According to one embodiment, the target RNA of interest comprises aproduct of a gene of the eukaryotic cell conferring resistance to thepathogen (e.g. virus, bacteria, fungi, etc.). Exemplary genes include,but are not limited to, CyPA-(Cyclophilins (CyPs)), Cyclophilin A (e.g.for Hepatitis C virus infection), CD81, scavenger receptor class B typeI (SR-BI), ubiquitin specific peptidase 18 (USP18), phosphatidylinositol4-kinase III alpha (PI4K-IIIα) (e.g. for HSV infection) and CCR5—(e.g.for HIV infection).According to one embodiment, the target RNA ofinterest comprises a product of a gene of the pathogen.

According to one embodiment, the virus is an arbovirus (e.g. Vesicularstomatitis Indiana virus—VSV). According to one embodiment, the targetRNA of interest comprises a product of a VSV gene, e.g. G protein (G),large protein (L), phosphoprotein, matrix protein (M) or nucleoprotein.

According to one embodiment, the target RNA of interest includes but isnot limited to gag and/or vif genes (i.e. conserved sequences in HIV-1);P protein (i.e. an essential subunit of the viral RNA-dependent RNApolymerase in RSV); P mRNA (i.e. in PIV); core, NS3, NS4B and NS5B (i.e.in HCV); VAMP-associated protein (hVAP-A), La antigen and polypyrimidinetract binding protein (PTB) (i.e. for HCV).

According to a specific embodiment, when the organism is a human, thetarget RNA of interest includes, but is not limited to, a gene of apathogen causing Malaria; a gene of HIV virus (e.g. as set forth inGenBank Accession No: NC_001802.1); a gene of HCV virus (e.g. as setforth in GenBank Accession No: NC_004102.1); and a gene of Parasiticworms (e.g. as set forth in GenBank Accession No: XM_003371604.1).

According to a specific embodiment, when the organism is a human, thetarget RNA of interest includes, but is not limited to, a gene relatedto a cancerous disease (e.g. Homo sapiens mRNA for bcr/abl e8a2 fusionprotein, as set forth in GenBank Accession No: AB069693.1) or a generelated to a myelodysplastic syndrome (MDS) and to vascular diseases(e.g. Human heparin-binding vascular endothelial growth factor (VEGF)mRNA, as set forth in GenBank Accession No: M32977.1).

According to a specific embodiment, when the organism is a cattle, thetarget RNA of interest includes, but is not limited to, a gene ofInfectious bovine rhinotracheitis virus (e.g. as set forth in GenBankAccession No: AJ004801.1), a type 1 bovine herpesvirus (BHV1), causinge.g. BRD (Bovine Respiratory Disease complex); a gene of Bluetonguedisease (BTV virus) (e.g. as set forth in GenBank Accession No:KP821170.1); a gene of Bovine Virus Diarrhhoea (BVD) (e.g. as set forthin GenBank Accession No: NC_001461.1); a gene of picornavirus (e.g. asset forth in GenBank Accession No: NC_004004.1), causing e.g. Foot &Mouth disease; a gene of Parainfluenza virus type 3 (PI3) (e.g. as setforth in GenBank Accession No: NC_028362.1), causing e.g. BRD; a gene ofMycobacterium bovis (M. bovis) (e.g. as set forth in GenBank AccessionNo: NC 037343.1), causing e.g. Bovine Tuberculosis (bTB).

According to a specific embodiment, when the organism is a sheep, thetarget RNA of interest includes, but is not limited to, a gene of apathogen causing Tapeworms disease (E. granulosus life cycle,Echinococcus granulosus, Taenia ovis, Taenia hydatigena, Monieziaspecies) (e.g. as set forth in GenBank Accession No: AJ012663.1); a geneof a pathogen causing Flatworms disease (Fasciola hepatica, Fasciolagigantica, Fascioloides magna, Dicrocoelium dendriticum, Schistosomabovis) (e.g. as set forth in GenBank Accession No: AY644459.1); a geneof a pathogen causing Bluetongue disease (BTV virus, e.g. as set forthin GenBank Accession No: KP821170.1); and a gene of a pathogen causingRoundworms disease (Parasitic bronchitis, also known as ““hoose””,Elaeophora schneideri, Haemonchus contortus, Trichostrongylus species,Teladorsagia circumcincta, Cooperia species, Nematodirus species,Dictyocaulus filaria, Protostrongylus refescens, Muellerius capillaris,Oesophagostomum species, Neostrongylus linearis, Chabertia ovina,Trichuris ovis) (e.g. as set forth in GenBank Accession No:NC_003283.11).

According to a specific embodiment, when the organism is a pig, thetarget RNA of interest includes, but is not limited to, a gene ofAfrican swine fever virus (ASFV) (causing e.g. African Swine Fever)(e.g. as set forth in GenBank Accession No: NC_001659.2); a gene ofClassical swine fever virus (causing e.g. Classical Swine Fever) (e.g.as set forth in GenBank Accession No: NC_002657.1); and a gene of apicornavirus (causing e.g. Foot & Mouth disease) (e.g. as set forth inGenBank Accession No: NC_004004.1).

According to a specific embodiment, when the organism is a chicken, thetarget RNA of interest includes, but is not limited to, a gene of Birdflu (or Avian influenza), a gene of a variant of avian paramyxovirus 1(APMV-1) (causing e.g. Newcastle disease), or a gene of a pathogencausing Marek's disease.

According to a specific embodiment, when the organism is a tadpoleshrimp, the target RNA of interest includes, but is not limited to, agene of White Spot Syndrome Virus (WSSV), a gene of Yellow Head Virus(YHV), or a gene of Taura Syndrome Virus (TSV).

According to a specific embodiment, when the organism is a salmon, thetarget RNA of interest includes, but is not limited to, a gene ofInfectious Salmon Anaemia (ISA), a gene of Infectious HematopoieticNecrosis (IHN), a gene of Sea lice (e.g. ectoparasitic copepods of thegenera Lepeophtheirus and Caligus).

Exemplary endogenous non-coding RNA molecules which may be modified totarget the RNA of interest (e.g. a gene of a pathogen), exemplarysequences of gRNA (i.e. a DNA editing agent) which may be used to modifythe endogenous non-coding RNA molecules, and exemplary nucleotidesequences for redirecting a silencing specificity of the endogenousnon-coding RNA molecule towards the target RNA of interest are providedin Table 1B, hereinbelow.

TABLE 1B Examples of GEiGS oligo designs to generate different traits invarious hosts oligo_seq pam_difference Sequence of GEiGS seq Number ofoligo, consisting difference sg_seq nucleotide of the precursor from wtSequence of changes sequence with its Number of the CRISPR/ between thecorresponding nucleotide cas9 small wild type mature replaced changesguide RNA precursor and by a siRNA between the targetting the the GEiGStargeting wild type precursor sequence that Host, trait andmiRNA-template the desired precursor sequence for fall in the Host(bold); molecule - and the swapping - PAM region pathogen/pest/disease(italics) Oligo info SEQ ID NO: GeiGs oligo SEQ ID NO: of the sgRNAsgRNA_strand Bos taurus (Cattle) AJ004801.1/Infectious bovinerhinotracheitis virus, a type 1 bovine herpesvirus (BHV1) causing BRD(Bovine Respiratory Disease complex) bta-mir-222 Max change/ 93 63 165 3fw perfect structure/ trait-specific siRNA bta-mir-484 Min change/ 94 14166 3 rv perfect structure/ trait-specific siRNA bta-mir-222 Max change/95 54 167 3 fw altered structure/ trait-specific siRNA bta-mir-127 Maxchange/ 96 58 168 3 rv perfect structure/ non-specific siRNAKP821170.1/Bluetongue disease (BTV virus) bta-mir-222 Max change/ 97 64169 3 fw perfect structure/ trait-specific siRNA bta-mir-484 Min change/98 18 170 3 rv perfect structure/ trait-specific siRNA bta-mir-222 Maxchange/ 99 53 171 3 fw altered structure/ trait-specific siRNAbta-mir-221 Max change/ 100 59 172 3 fw perfect structure/ non-specificsiRNA NC_001461.1/Bovine Virus Diarrhhoea (BVD) bta-mir-222 Max change/101 65 173 3 fw perfect structure/ trait-specific siRNA bta-mir-215 Minchange/ 102 15 174 3 rv perfect structure/ trait-specific siRNAbta-mir-222 Max change/ 103 45 175 3 fw altered structure/trait-specific siRNA bta-mir-99a Max change/ 104 58 176 3 fw perfectstructure/ non-specific siRNA NC_004004.1/Foot & Mouth disease (a viraldisease caused by a picornavirus) bta-mir-222 Max change/ 105 65 177 3fw perfect structure/ trait-specific siRNA bta-mir-484 Min change/ 10618 178 3 rv perfect structure/ trait-specific siRNA bta-mir-125a Maxchange/ 107 48 179 3 rv altered structure/ trait-specific siRNAbta-mir-127 Max change/ 108 63 180 3 rv perfect structure/ non-specificsiRNA NC_028362.1/Parainfluenza virus type 3 (PI3) causing BRDbta-mir-222 Max change/ 109 64 181 3 fw perfect structure/trait-specific siRNA bta-mir-126 Min change/ 110 19 182 3 rv perfectstructure/ trait-specific siRNA bta-mir-125a Max change/ 111 49 183 3 rvaltered structure/ trait-specific siRNA bta-mir-31 Max change/ 112 60184 2 rv perfect structure/ non-specific siRNA NC_037343.1/BovineTuberculosis (bTB) caused by the bacterium Mycobacterium bovis (M.bovis) bta-mir-222 Max change/ 113 64 185 3 fw perfect structure/trait-specific siRNA bta-mir-26b Min change/ 114 19 186 3 fw perfectstructure/ trait-specific siRNA bta-mir-125a Max change/ 115 36 187 2 rvaltered structure/ trait-specific siRNA bta-mir-127 Max change/ 116 60188 3 rv perfect structure/ non-specific siRNA Homo sapiens (Human)AB069693.1/Homo sapiens mRNA for bcr/abl e8a2 fusion protein hsa-mir-98Max change/ 117 65 189 1 fw perfect structure/ trait-specific siRNAhsa-mir-30a Min change/ 118 14 190 3 fw perfect structure/trait-specific siRNA hsa-mir-100 Max change/ 119 38 191 2 fw alteredstructure/ trait-specific siRNA hsa-mir-98 Max change/ 120 61 192 3 fwperfect structure/ non-specific siRNA M32977.1/Human heparin-bindingvascular endothelial growth factor (VEGF) mRNA hsa-mir-98 Max change/121 64 193 1 fw perfect structure/ trait-specific siRNA hsa-mir-24-2 Minchange/ 122 16 194 3 rv perfect structure/ trait-specific siRNAhsa-mir-100 Max change/ 123 35 195 2 fw altered structure/trait-specific siRNA hsa-mir-98 Max change/ 124 62 196 1 fw perfectstructure/ non-specific siRNA NC_001802.1/HIV virus hsa-mir-98 Maxchange/ 125 62 197 1 fw perfect structure/ trait-specific siRNAhsa-mir-26b Min change/ 126 15 198 1 rv perfect structure/trait-specific siRNA hsa-mir-100 Max change/ 127 32 199 1 fw alteredstructure/ trait-specific siRNA hsa-mir-19b-l Max change/ 128 60 200 3rv perfect structure/ non-specific siRNA NC_004102.1/HCV virushsa-mir-98 Max change/ 129 62 201 3 fw perfect structure/ trait-specificsiRNA hsa-mir-20a Min change/ 130 16 202 3 fw perfect structure/trait-specific siRNA hsa-let-7c Max change/ 131 39 203 3 fw alteredstructure/ trait-specific siRNA hsa-mir-98 Max change/ 132 62 204 1 fwperfect structure/ non-specific siRNA XM_003371604.1/Parasitic wormshsa-mir-98 Max change/ 133 66 205 2 fw perfect structure/ trait-specificsiRNA hsa-mir-20a Min change/ 134 17 206 3 fw perfect structure/trait-specific siRNA hsa-mir-100 Max change/ 135 42 207 1 fw alteredstructure/ trait-specific siRNA hsa-mir-98 Max change/ 136 61 208 1 fwperfect structure/ non-specific siRNA Ovis aries (Sheep)AJ012663.1/Tapeworms disease (E. granulosus life cycle, Echinococcusgranulosus, Taenia ovis, Taenia hydatigena, Moniezia species)oar-mir-127 Max change/ 137 75 209 1 fw perfect structure/trait-specific siRNA oar-mir-125b Min change/ 138 19 210 3 rv perfectstructure/ trait-specific siRNA oar-mir-411a Max change/ 139 65 211 3 rvaltered structure/ trait-specific siRNA oar-mir-382 Max change/ 140 78212 3 fw perfect structure/ non-specific siRNA AY644459.1/Flatwormsdisease (Fasciola hepatica, Fasciola gigantica, Fascioloides magna,Dicrocoelium dendriticum, Schistosoma bovis) oar-mir-382 Max change/ 14173 213 3 fw perfect structure/ trait-specific siRNA oar-mir-134 Minchange/ 142 20 214 3 fw perfect structure/ trait-specific siRNAoar-mir-543 Max change/ 143 42 215 3 rv altered structure/trait-specific siRNA oar-mir-382 Max change/ 144 76 216 3 fw perfectstructure/ non-specific siRNA KP821170.1/Bluetongue disease (BTV virus)oar-mir-382 Max change/ 145 76 217 3 fw perfect structure/trait-specific siRNA oar-mir-134 Min change/ 146 20 218 3 fw perfectstructure/ trait-specific siRNA oar-mir-411a Max change/ 147 50 219 3 rvaltered structure/ trait-specific siRNA oar-mir-382 Max change/ 148 75220 3 fw perfect structure/ non-specific siRNA NC_003283.11/Roundwormsdisease (Parasitic bronchitis, also known as ““hoose””, Elaeophoraschneideri, Haemonchus contortus, Trichostrongylus species, Teladorsagiacircumcincta, Cooperia species, Nematodirus species, Dictyocaulusfilaria, Protostrongylus refescens, Muellerius capillaris,Oesophagostomum species, Neostrongylus linearis, Chabertia ovina,Trichuris ovis) oar-mir-382 Max change/ 149 75 221 3 fw perfectstructure/ trait-specific siRNA oar-mir-665 Min change/ 150 14 222 3 rvperfect structure/ trait-specific siRNA oar-mir-379 Max change/ 151 52223 1 rv altered structure/ trait-specific siRNA oar-mir-411a Maxchange/ 152 78 224 3 rv perfect structure/ non-specific siRNA Sus scrofa(Pig) NC_001659.2/African Swine Fever (disease caused by African swinefever virus (ASFV)) ssc-mir-186 Max change/ 153 60 225 3 rv perfectstructure/ trait-specific siRNA ssc-mir-122 Min change/ 154 20 226 2 fwperfect structure/ trait-specific siRNA ssc-mir-204 Max change/ 155 46227 3 rv altered structure/ trait-specific siRNA ssc-mir-15b Max change/156 60 228 3 fw perfect structure/ non-specific siRNANC_002657.1/Classical Swine Fever (caused by a small RNA virus with alipid envelope, the Classical swine fever virus- influenza virus)ssc-mir-204 Max change/ 157 61 229 3 rv perfect structure/trait-specific siRNA ssc-mir-122 Min change/ 158 19 230 3 fw perfectstructure/ trait-specific siRNA ssc-mir-204 Max change/ 159 54 231 3 rvaltered structure/ trait-specific siRNA ssc-mir-186 Max change/ 160 59232 2 rv perfect structure/ non-specific siRNA NC_004004.1/Foot & Mouthdisease (a viral disease caused by a picornavirus) ssc-mir-325 Maxchange/ 161 60 233 1 rv perfect structure/ trait-specific siRNAssc-mir-122 Min change/ 162 19 234 2 fw perfect structure/trait-specific siRNA ssc-mir-204 Max change/ 163 46 235 3 rv alteredstructure/ trait-specific siRNA ssc-mir-21 Max change/ 164 60 236 3 fwperfect structure/ non-specific siRNA Table 1B provides example GEiGSoligos designed against a variety of targets in several host organisms.For each host-target combination, four oligos are provided: minimumsequence changes with matching structure and efficient siRNA; maximumsequence changes with matching structure and efficient siRNA; maximumsequence changes and non-matching structure and efficient siRNA; andmaximum sequence changes with matching structure and inefficient siRNA.

Assessing the efficacy of treatment may be carried out using any methodknown in the art, such as by assessing the subject's physicalwell-being, by blood tests, by assessing viral/bacterial load, etc.

According to one aspect of the invention, there is provided a method oftreating a monogenic recessive disorder in a subject in need thereof,the method comprising modifying a gene encoding or processed into anon-coding RNA molecule or encoding or processed into an RNA silencingmolecule according to the method of some embodiments of the invention,wherein the target RNA of interest is associated with the monogenicrecessive disorder, thereby treating the monogenic recessive disorder inthe subject.

According to one aspect of the invention, there is provided a DNAediting agent conferring a silencing specificity of a non-coding RNAmolecule having no RNA silencing activity towards a target RNA ofinterest, wherein the target RNA of interest is associated with amonogenic recessive disorder, for use in treating a monogenic recessivedisorder in a subject in need thereof.

According to one aspect of the invention, there is provided a DNAediting agent redirecting a silencing specificity of a gene encoding orprocessed into a RNA silencing molecule to a target RNA towards a secondtarget RNA, the target RNA and the second target RNA being distinct,wherein the second target RNA is associated with a monogenic recessivedisorder, for use in treating a monogenic recessive disorder in asubject in need thereof.

As used herein, the term “monogenic recessive disorder” refers to adisease or condition caused as a result of a single defective gene onthe autosomes.

According to one embodiment, the monogenic recessive disorder is aresult of a spontaneous or hereditary mutation.

According to one embodiment, the monogenic recessive disorder isautosomal dominant, autosomal recessive or X-linked recessive.

Exemplary monogenic recessive disorders include, but are not limited to,severe combined immunodeficiency (SCID), hemophilia, enzymedeficiencies, Parkinson's Disease, Wiskott-Aldrich syndrome, CysticFibrosis, Phenylketonuria, Friedrich's Ataxia, Duchenne MuscularDystrophy, Hunter disease, Aicardi Syndrome, Klinefelter's Syndrome,Leber's hereditary optic neuropathy (LHON).

According to one embodiment, in order to treat a monogenic recessivedisorder in a subject, the non-coding RNA molecule (e.g. RNA silencingmolecule) is designed to target a RNA of interest associated with themonogenic recessive disorder.

According to one embodiment, when the disorder is Parkinson's diseasethe target RNA of interest comprises a product of a SNCA (PARK1=4),LRRK2 (PARKS), Parkin (PARK2), PINK1 (PARK6), DJ-1 (PARK7), or ATP13A2(PARK9) gene.

According to one embodiment, when the disorder is hemophilia or vonWillebrand disease the target RNA of interest comprises, for example, aproduct of an anti-thrombin gene, of coagulation factor VIII gene or offactor IX gene.

Assessing the efficacy of treatment may be carried out using any methodknown in the art, such as by assessing the subject's physicalwell-being, by blood tests, bone marrow aspirate, etc.

According to one aspect of the invention, there is provided a method oftreating an autoimmune disease in a subject in need thereof, the methodcomprising modifying a gene encoding or processed into a non-coding RNAmolecule or encoding or processed into an RNA silencing moleculeaccording to the method of some embodiments of the invention, whereinthe target RNA of interest is associated with the autoimmune disease,thereby treating the autoimmune disease in the subject.

According to one aspect of the invention, there is provided a DNAediting agent conferring a silencing specificity of a non-coding RNAmolecule having no RNA silencing activity towards a target RNA ofinterest, wherein the target RNA of interest is associated with anautoimmune disease, for use in treating an autoimmune disease in asubject in need thereof.

According to one aspect of the invention, there is provided a DNAediting agent redirecting a silencing specificity of a gene encoding orprocessed into a RNA silencing molecule to a target RNA towards a secondtarget RNA, the target RNA and the second target RNA being distinct,wherein the second target RNA is associated with an autoimmune disease,for use in treating an autoimmune disease in a subject in need thereof.

Non-limiting examples of autoimmune diseases include, but are notlimited to, cardiovascular diseases, rheumatoid diseases, glandulardiseases, gastrointestinal diseases, cutaneous diseases, hepaticdiseases, neurological diseases, muscular diseases, nephric diseases,diseases related to reproduction, connective tissue diseases andsystemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660), anti-factorVIII autoimmune disease (Lacroix-Desmazes S. et al., Semin ThrombHemost.2000; 26 (2):157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne(Paris). 2000 May; 151 (3):178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999; 14 (4):171), antibody-induced heartfailure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17; 83 (12A):75H),thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 April-June;14 (2):114; Semple J W. et al., Blood 1996 May 15; 87 (10):4245),autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March; 74(3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al.,J Clin Invest 1996 October 15; 98 (8):1709) and anti-helper T lymphocyteautoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11 (1):9).Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July;15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res ClinPract 1996 October; 34 Suppl:S125), autoimmune thyroid diseases, Graves'disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29(2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77),spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al.,Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema (Mitsuma T.Nippon Rinsho. 1999 August; 57 (8):1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 February; 37 (2):87), autoimmuneanti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000March; 43 (3):134), autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893) and Type I autoimmune polyglandularsyndrome (Hara T. et al., Blood. 1991 Mar. 1; 77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) andautoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E,Int Rev Immunol 1999; 18 (1-2):83; Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), neuropathies, motor neuropathies (Kornberg A J.J Clin Neurosci. 2000 May; 7 (3):191); Guillain-Barre syndrome andautoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April; 319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. AmJ Med Sci. 2000 April; 319 (4):204); paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy andstiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units S A2001 Mar. 27; 98 (7):3988); non-paraneoplastic stiff man syndrome,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C.and Honnorat J. Rev Neurol (Paris) 2000 January; 156 (1):23); dysimmuneneuropathies (Nobile-Orazio E. et al., Electroencephalogr ClinNeurophysiol Suppl 1999; 50:419); acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J NeurolNeurosurg Psychiatry 1994 May; 57 (5):544) and neurodegenerativediseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother1999 June; 53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol 1990 August; 1 (2):140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7Suppl 2:S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol 1994 August; 157 (1):249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107).

According to one embodiment, the autoimmune disease comprises systemiclupus erythematosus (SLE).

According to one embodiment, in order to treat an autoimmune disease ina subject, the non-coding RNA molecule (e.g. RNA silencing molecule) isdesigned to target a RNA of interest associated with the autoimmunedisease.

According to one embodiment, when the disease is lupus, the target RNAof interest comprises an antinuclear antibody (ANA) such as thatpathologically produced by B cells.

Assessing the efficacy of treatment may be carried out using any methodknown in the art, such as by assessing the subject's physicalwell-being, by blood tests, bone marrow aspirate, etc.

According to one aspect of the invention, there is provided a method oftreating a cancerous disease in a subject in need thereof, the methodcomprising modifying a gene encoding or processed into a non-coding RNAmolecule or encoding or processed into an RNA silencing moleculeaccording to the method of some embodiments of the invention, whereinthe target RNA of interest is associated with the cancerous disease,thereby treating the cancerous disease in the subject.

According to one aspect of the invention, there is provided a DNAediting agent conferring a silencing specificity of a non-coding RNAmolecule having no RNA silencing activity towards a target RNA ofinterest, wherein the target RNA of interest is associated with acancerous disease, for use in treating a cancerous disease in a subjectin need thereof.

According to one aspect of the invention, there is provided a DNAediting agent redirecting a silencing specificity of a gene encoding orprocessed into a RNA silencing molecule to a target RNA towards a secondtarget RNA, the target RNA and the second target RNA being distinct,wherein the second target RNA is associated with a cancerous disease,for use in treating a cancerous disease in a subject in need thereof.

Non-limiting examples of cancers which can be treated by the method ofsome embodiments of the invention can be any solid or non-solid cancerand/or cancer metastasis or precancer, including, but is not limitingto, tumors of the gastrointestinal tract (colon carcinoma, rectalcarcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma,hereditary nonpolyposis type 1, hereditary nonpolyposis type 2,hereditary nonpolyposis type 3, hereditary nonpolyposis type 6;colorectal cancer, hereditary nonpolyposis type 7, small and/or largebowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer,stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors),endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladdercarcinoma, Biliary tract tumors, prostate cancer, prostateadenocarcinoma, renal cancer (e.g., Wilms' tumor type 2 or type 1),liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma,hepatocellular cancer), bladder cancer, embryonal rhabdomyosarcoma, germcell tumor, trophoblastic tumor, testicular germ cells tumor, immatureteratoma of ovary, uterine, epithelial ovarian, sacrococcygeal tumor,choriocarcinoma, placental site trophoblastic tumor, epithelial adulttumor, ovarian carcinoma, serous ovarian cancer, ovarian sex cordtumors, cervical carcinoma, uterine cervix carcinoma, small-cell andnon-small cell lung carcinoma, nasopharyngeal, breast carcinoma (e.g.,ductal breast cancer, invasive intraductal breast cancer, sporadic;breast cancer, susceptibility to breast cancer, type 4 breast cancer,breast cancer-1, breast cancer-3; breast-ovarian cancer), squamous cellcarcinoma (e.g., in head and neck), neurogenic tumor, astrocytoma,ganglioblastoma, neuroblastoma, lymphomas (e.g., Hodgkin's disease,non-Hodgkin's lymphoma, B cell, Burkitt, cutaneous T cell, histiocytic,lymphoblastic, T cell, thymic), gliomas, adenocarcinoma, adrenal tumor,hereditary adrenocortical carcinoma, brain malignancy (tumor), variousother carcinomas (e.g., bronchogenic large cell, ductal, Ehrlich-Lettreascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid,oat cell, small cell, spindle cell, spinocellular, transitional cell,undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma),ependimoblastoma, epithelioma, erythroleukemia (e.g., Friend,lymphoblast), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma(e.g., multiforme, astrocytoma), glioma hepatoma, heterohybridoma,heteromyeloma, histiocytoma, hybridoma (e.g., B cell), hypernephroma,insulinoma, islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma,leukemia (e.g., acute lymphatic, acute lymphoblastic, acutelymphoblastic pre-B cell, acute lymphoblastic T cell leukemia,acute—megakaryoblastic, monocytic, acute myelogenous, acute myeloid,acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid,chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairycell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage,myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell,promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition tomyeloid malignancy, acute nonlymphocytic leukemia), lymphosarcoma,melanoma, mammary tumor, mastocytoma, medulloblastoma, mesothelioma,metastatic tumor, monocyte tumor, multiple myeloma, myelodysplasticsyndrome, myeloma, nephroblastoma, nervous tissue glial tumor, nervoustissue neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma,osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), papilloma,transitional cell, pheochromocytoma, pituitary tumor (invasive),plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's,histiocytic cell, Jensen, osteogenic, reticulum cell), schwannoma,subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma,testicular tumor, thymoma and trichoepithelioma, gastric cancer,fibrosarcoma, glioblastoma multiforme; multiple glomus tumors,Li-Fraumeni syndrome, liposarcoma, lynch cancer family syndrome II, malegerm cell tumor, mast cell leukemia, medullary thyroid, multiplemeningioma, endocrine neoplasia myxosarcoma, paraganglioma, familialnonchromaffin, pilomatricoma, papillary, familial and sporadic, rhabdoidpredisposition syndrome, familial, rhabdoid tumors, soft tissue sarcoma,and Turcot syndrome with glioblastoma.

According to one embodiment, the cancer which can be treated by themethod of some embodiments of the invention comprises a hematologicmalignancy. An exemplary hematologic malignancy comprises one whichinvolves malignant fusion of the ABL tyrosine kinase to different otherchromosomes generating what is termed BCR-ABL which in turn resulting inmalignant fusion protein. Accordingly, targeting the fusion point in themRNA may silence only the fusion mRNA for down-regulation while thenormal proteins, essential for the cell, will be, spared.

According to one embodiment, in order to treat a cancerous disease in asubject, the non-coding RNA molecule (e.g. RNA silencing molecule) isdesigned to target a RNA of interest associated with the cancerousdisease.

According to one embodiment, the target RNA of interest comprises aproduct of an oncogene (e.g. mutated oncogene).

According to one embodiment, the target RNA of interest restores thefunction of a tumor suppressor.

According to one embodiment, the target RNA of interest comprises aproduct of a RAS, MCL-1 or MYC gene.

According to one embodiment, the target RNA of interest comprises aproduct of a BCL-2 family of apoptosis-related genes.

Exemplary target genes include, but are not limited to, mutant dominantnegative TP53, Bcl-x, IAPs, Flip, Faim3 and SMS 1.

According to one embodiment, when the cancer is melanoma, the target RNAof interest comprises BRAF. Several forms of BRAF mutations arecontemplated herein, including e.g. V600E, V600K, V600D, V600G, andV600R.

According to one embodiment, the method is affected by targetingnon-coding RNA molecules in healthy immune cells, such as white bloodcells e.g. T cells, B cells or NK cells (e.g. from a patient or from acell donor) to a target a RNA of interest such that the immune cells arecapable of killing (directly or indirectly) malignant cells (e.g. cellsof a hematological malignancy).

According to one embodiment, the method is affected by targetingnon-coding RNA molecules to silence proteins (i.e. target RNA ofinterest) that are manipulated by cancer factors (i.e. in order tosuppress immune responses from recognizing the malignancy), such thatthe cancer can be recognized and eradicated by the native immune system.

Assessing the efficacy of treatment may be carried out using any methodknown in the art, such as by assessing the tumor growth or the number ofneoplasms or metastases, e.g. by MRI, CT, PET-CT, by blood tests,ultrasound, x-ray, etc.

According to one aspect of the invention, there is provided a method ofenhancing efficacy and/or specificity of a chemotherapeutic agent in asubject in need thereof, the method comprising modifying a gene encodingor processed into a non-coding RNA molecule or encoding or processedinto an RNA silencing molecule according to the method of someembodiments of the invention, wherein the target RNA of interest isassociated with enhancement of efficacy and/or specificity of thechemotherapeutic agent, thereby enhancing efficacy and/or specificity ofa chemotherapeutic agent in the subject.

According to one aspect of the invention, there is provided a DNAediting agent conferring a silencing specificity of a non-coding RNAmolecule having no RNA silencing activity towards a target RNA ofinterest, wherein the target RNA of interest is associated with anenhancement of efficacy and/or specificity of the chemotherapeuticagent, for use in enhancing efficacy and/or specificity of achemotherapeutic agent in a subject in need thereof.

According to one aspect of the invention, there is provided a DNAediting agent redirecting a silencing specificity of a gene encoding orprocessed into a RNA silencing molecule to a target RNA towards a secondtarget RNA, the target RNA and the second target RNA being distinct,wherein the second target RNA is associated with an enhancement ofefficacy and/or specificity of the chemotherapeutic agent, for use inenhancing efficacy and/or specificity of a chemotherapeutic agent in asubject in need thereof.

As used herein, the term “chemotherapeutic agent” refer to an agent thatreduces, prevents, mitigates, limits, and/or delays the growth ofneoplasms or metastases, or kills neoplastic cells directly by necrosisor apoptosis of neoplasms or any other mechanism, or that can beotherwise used, in a pharmaceutically-effective amount, to reduce,prevent, mitigate, limit, and/or delay the growth of neoplasms ormetastases in a subject with neoplastic disease (e.g. cancer).

Chemotherapeutic agents include, but are not limited to,fluoropyrimidines; pyrimidine nucleosides; purine nucleosides;anti-folates, platinum agents; anthracyclines/anthracenediones;epipodophyllotoxins; camptothecins (e.g., Karenitecin); hormones;hormonal complexes; antihormonals; enzymes, proteins, peptides andpolyclonal and/or monoclonal antibodies;

immunological agents; vinca alkaloids; taxanes; epothilones;antimicrotubule agents; alkylating agents; antimetabolites;topoisomerase inhibitors; antivirals; and various other cytotoxic andcytostatic agents.

According to a specific embodiment, the chemotherapeutic agent includes,but is not limited to, abarelix, aldesleukin, aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenictrioxide, asparaginase, azacitidine, bevacuzimab, bexarotene, bleomycin,bortezomib, busulfan, calusterone, capecitabine, carboplatin,carmustine, celecoxib, cetuximab, cisplatin, cladribine, clofarabine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, actinomycin D,Darbepoetin alfa, Darbepoetin alfa, daunorubicin liposomal,daunorubicin, decitabine, Denileukindiftitox, dexrazoxane, dexrazoxane,docetaxel, doxorubicin, dromostanolone propionate, Elliott's B Solution,epirubicin, Epoetin alfa, erlotinib, estramustine, etoposide,exemestane, Filgrastim, floxuridine, fludarabine, fluorouracil 5-FU,fulvestrant, gefitinib, gemcitabine, gemtuzumabozogamicin, goserelinacetate, histrelin acetate, hydroxyurea, IbritumomabTiuxetan,idarubicin, ifosfamide, imatinibmesylate, interferon alfa 2a, Interferonalfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, LeuprolideAcetate, levamisole, lomustine, CCNU, meclorethamine, nitrogen mustard,megestrol acetate, melphalan, L-PAM, mercaptopurine 6-MP, mesna,methotrexate, mitomycin C, mitotane, mitoxantrone,nandrolonephenpropionate, nelarabine, Nofetumomab, Oprelvekin,Oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate,pegademase, pegaspargase, Pegfilgrastim, pemetrexed disodium,pentostatin, pipobroman, plicamycinmithramycin, porfimer sodium,procarbazine, quinacrine, Rasburicase, Rituximab, sargramostim,sorafenib, streptozocin, sunitinib maleate, tamoxifen, temozolomide,teniposide VM-26, testolactone, thioguanine 6-TG, thiotepa, thiotepa,topotecan, toremifene, Tositumomab, Trastuzumab, tretinoin ATRA, UracilMustard, valrubicin, vinblastine, vinorelbine, zoledronate andzoledronic acid.

According to one embodiment, the effect of the chemotherapeutic agent isenhanced by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99% or by 100% as compared to the effect of a chemotherapeutic agent ina subject not treated by the DNA editing agent designed to confer asilencing activity and/or specificity of a non-coding RNA molecule (e.g.RNA silencing molecule) towards a target RNA of interest.

Assessing the efficacy and/or specificity of a chemotherapeutic agentmay be carried out using any method known in the art, such as byassessing the tumor growth or the number of neoplasms or metastases,e.g. by MRI, CT, PET-CT, by blood tests, ultrasound, x-ray, etc.

According to one embodiment, the method is affected by targetingnon-coding RNA molecules in healthy immune cells, such as white bloodcells e.g. T cells, B cells or NK cells (e.g. from a patient or from acell donor) to target a RNA of interest such that the immune cells arecapable of decreasing resistance of the cancer to chemotherapy.

According to one embodiment, the method is affected by targetingnon-coding RNA molecules in healthy immune cells, such as white bloodcells e.g. T cells, B cells or NK cells (e.g. from a patient or from acell donor) to target a RNA of interest such that the immune cells areresistant to chemotherapy.

According to one embodiment, in order to enhance efficacy and/orspecificity of a chemotherapeutic agent in a subject, the non-coding RNAmolecule (e.g. RNA silencing molecule) is designed to target a RNA ofinterest associated with suppression of efficacy and/or specificity ofthe chemotherapeutic agent.

According to one embodiment, the target RNA of interest comprises aproduct of a drug-metabolising enzyme gene (e.g. cytochrome P450 [CYP]2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, dihydropyrimidinedehydrogenase, uridine diphosphate glucuronosyltransferase [UGT] 1A1,glutathione S-transferase, sulfotransferase [SULT] 1A1,N-acetyltransferase [NAT], thiopurine methyltransferase [TPMT]) and drugtransporters (P-glycoprotein [multidrug resistance 1], multidrugresistance protein 2 [MRP2], breast cancer resistance protein [BCRP]).

According to one embodiment, the target RNA of interest comprises ananti-apoptotic gene. Exemplary target genes include, but are not limitedto, Bcl-2 family members, e.g. Bcl-x, IAPs, Flip, Faim3 and SMS1.

According to one aspect of the invention, there is provided a method ofinducing cell apoptosis in a subject in need thereof, the methodcomprising modifying a gene encoding or processed into a non-coding RNAmolecule or encoding or processed into an RNA silencing moleculeaccording to the method of some embodiments of the invention, whereinthe target RNA of interest is associated with apoptosis, therebyinducing cell apoptosis in the subject.

According to one aspect of the invention, there is provided a DNAediting agent conferring a silencing specificity of a non-coding RNAmolecule having no RNA silencing activity towards a target RNA ofinterest, wherein the target RNA of interest is associated withapoptosis, for use in inducing cell apoptosis in a subject in needthereof.

According to one aspect of the invention, there is provided a DNAediting agent redirecting a silencing specificity of a gene encoding orprocessed into a RNA silencing molecule to a target RNA towards a secondtarget RNA, the target RNA and the second target RNA being distinct,wherein the second target RNA is associated with apoptosis, for use ininducing cell apoptosis in a subject in need thereof.

The term “cell apoptosis” as used herein refers to the cell process ofprogrammed cell death. Apoptosis characterized by distinct morphologicalterations in the cytoplasm and nucleus, chromatin cleavage atregularly spaced sites, and endonucleolytic cleavage of genomic DNA atinternucleosomal sites. These changes include blebbing, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomal DNAfragmentation.

According to one embodiment, cell apoptosis is enhanced by about 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or by 100% ascompared to cell apoptosis in a subject not treated by the DNA editingagent conferring a silencing activity and/or specificity of a non-codingRNA molecule (e.g. RNA silencing molecule) towards a target RNA ofinterest.

Assessing cell apoptosis may be carried out using any method known inthe art, e.g. cell proliferation assay, FACS analysis etc.

According to one embodiment, in order to induce cell apoptosis in asubject, the non-coding RNA molecule (e.g. RNA silencing molecule) isdesigned to target a RNA of interest associated with the apoptosis.

According to one embodiment, the target RNA of interest comprises aproduct of a BCL-2 family of apoptosis-related genes.

According to one embodiment, the target RNA of interest comprises ananti-apoptotic gene. Exemplary genes include, but are not limited to,mutant dominant negative TP53, Bcl-x, IAPs, Flip, Faim3 and SMS1.

According to one aspect of the invention, there is provided a method ofgenerating a eukaryotic non-human organism, with the proviso that theorganism is not a plant, wherein at least some of the cells of theeukaryotic non-human organism comprise a modified gene encoding orprocessed into a non-coding RNA molecule comprising a silencingspecificity of towards a target RNA of interest, the method comprisingintroducing into at least one cell of the eukaryotic non-human organisma DNA editing agent conferring a silencing specificity of the non-codingRNA molecule (e.g. RNA silencing molecule) towards the target RNA ofinterest.

The following information should be available: a) Target sequence to besilenced by Gene Editing induced Gene Silencing (GEiGS) (“target”); b)Choosing whether the GEiGS (i.e. the modified non-coding RNA) would beexpressed ubiquitously (e.g. constitutively) or specifically (e.g.expression specific to a certain tissue, developmental stage, stress,heat/cold shock etc.).

Submitting this information to publicly available miRNA datasets (e.g.small RNA sequencing, genomic sequences, microarrays etc.) so as tofilter (i.e. elect) only relevant miRNAs that match the input criteria:miRNAs that are expressed according to the requirement(s) describedabove.

Using publicly available tools, a list of potent target-specific siRNAsequences may be generated. The miRNAs may be aligned against the potentsiRNA sequences and the most homologous miRNAs may be elected. FilteredmiRNAs may have a similar sequence in the same orientation like thepotent siRNAs.

Modifying the naturally mature miRNAs sequences, which are scored tohave high homology to target-specific potent siRNAs, to perfectly matchthe target's sequence. This modification may occur in one mature miRNAstrand with the highest target homology (e.g. could be either theoriginal miRNA guide or passenger strand). Such 100% complementary tothe target can potentially turn the miRNA sequence into a siRNA.

Minimal GE may be achieved by filtering miRNA sequences with naturallyoccurring high homology (reverse complement) to the target.

Using the primary modified miRNA genes to generate ssDNA oligos (e.g.200-500 nt ssDNA long) and dsDNA fragments (e.g. 250-5000 nt dsDNAfragments only or cloned within plasmids) based on the genomic DNAsequences that flank the modified miRNA precursor sequence (pre-miRNA).The modified miRNA's guide strand (silencing strand) sequence may bedesigned to be 100% complementary to the target.

Modifying the sequence of the other miRNA gene region to preserve theoriginal (unmodified) miRNA precursor and mature structure, throughkeeping the same base pairing profile.

Designing sgRNAs to specifically target the original unmodified miRNAgene (specific to the genomic miRNA loci), and not the modified version(i.e. the oligo/fragment sequences).

Analyzing the comparative restriction enzyme site between the modifiedand the original miRNA gene and summarizing the differential restrictionsites. Such a detection system is based on PCR that is followed byrestriction enzyme digestion and gel electrophoresis.

Validating as discussed in detail above.

Examining the targeting of the non-coding RNA towards other targets(e.g. “off target effect”), using in silico methods, when the endogenousnon-coding RNA (e.g. miRNA) comprises naturally occurring high homologywith the target (e.g. 60-90%), so as to obtain specific silencing of thetarget of interest.

Minimally modifying the endogenous non-coding RNA (e.g. miRNA) to boostits potency to silence the target of interest.

Validating GEiGS outcome of the primary minimally edited miRNA genes togenerate candidate refined minimally edited miRNAs. An experimentallyeffective primary GEiGS outcome (the primary minimally edited miRNAgenes) is considered as a miRNA(s) with a guide or passenger strand thatis modified to match the target by 100%.

Generating several guide or passenger strand sequences that aregradually reverted back into the original sequence (as illustrated inFIG. 9).

Keeping the seed sequence in a way that there are at least 5 matches outof the seven seed nucleotides (nucleotides 2-8 from the 5′ terminus).

Testing the various candidate ‘refined minimally edited miRNA genes’ fortarget silencing efficiency. Choosing the gene GE-mediated knock-in thatprovides the highest silencing with the minimal miRNA sequencemodification.

Testing potential “off target effects” of refined minimally edited miRNAcandidates. A significant prediction for “off target effects” affectsthe final evaluation of the refined minimally edited miRNA genes.

Testing the less refined minimally edited miRNA gene candidates based onthe experimental validation.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

It is understood that any Sequence Identification Number (SEQ ID NO)disclosed in the instant application can refer to either a DNA sequenceor a RNA sequence, depending on the context where that SEQ ID NO ismentioned, even if that SEQ ID NO is expressed only in a DNA sequenceformat or a RNA sequence format. For example, SEQ ID NOs: 1-4 areexpressed in a DNA sequence format (e.g., reciting T for thymine), butit can refer to either a DNA sequence that corresponds to an gRNAnucleic acid sequence, or the RNA sequence of a RNA molecule nucleicacid sequence. Similarly, though some sequences are expressed in a RNAsequence format (e.g., reciting U for uracil), depending on the actualtype of molecule being described, it can refer to either the sequence ofa RNA molecule comprising a dsRNA, or the sequence of a DNA moleculethat corresponds to the RNA sequence shown. In any event, both DNA andRNA molecules having the sequences disclosed with any substitutes areenvisioned.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”,W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Experimental Procedures

Cell Culture

Tissue culture is carried out on human cell lines or in mouse embryonicstem cells. Human Bone Osteosarcoma Epithelial Cells (U20S), Humanretinal pigment epithelial cells (RPE1), Adenocarcinomic human alveolarbasal epithelial cells (A549), Cervical cancer cells (HeLa) or humancolorectal cancer cells (HCT116) are cultured in tissue culture mediumsupplemented with essential nutrients (amino acids, carbohydrates,vitamins, minerals), growth factors, hormones as needed. The cells arecultured in a CO₂ humidified incubator with controlled temperature (37°C.) under the appropriate physio-chemical conditions (pH buffer, osmoticpressure).

Survival Assay

Chemo-sensitivity is determined by crystal violet assay as previouslydescribed [Taniguchi et al., Cell (2002) 109: 459-72]. Cells are seededonto 12-well plates at 2×10⁴/well and treated with cisplatin,camptothecin (Sigma), paclitaxel (Sigma), AZD2281 (Axon Medchem) orNutlin3 (Selleckchem) at indicated doses. After incubation for 3 days,monolayers are fixed in 10% methanol containing 10% acetic acid.Adherent cells are stained with 0.5% crystal violet in methanol. Theabsorbed dye is resolubilized with methanol containing 0.1% SDS, whichis transferred into 96-well plates and measured photometrically (595 nm)in a microplate reader. Cell survival is calculated by normalizing theabsorbance to that of non-treated controls.

The same method as above can be scaled up to a 6 well plate format orlarger and then forming colonies are counted without resolubilizing thecrystal violet, this format is called clonogenic assay and is based onthe ability of the treated cells to grow into colony. Another assay thatis used is the metabolic activity-based cell viability assay XTT or anyother metabolic viability assay. XTT is a colorimetric assay used toassess cell viability as a function of cell number based on metabolicactivity. This rapid, sensitive, non-radioactive assay is detected usingstandard microplate absorbance readers. Cells are grown in a 96-wellplate at a density of 10⁴-10⁵ cells/well in 100 μL of culture mediumwith compounds to be tested and are cultured in a CO₂ incubator for24-48 hours. Fresh buffers are prepared each time before the assay: 10mM PMS solution in phosphate-buffered saline is and 4 mg of XTT isdissolved in 4 mL of 37° C. cell culture medium. 10 μL of the PMSsolution is added to 4 mL of XTT solution immediately before labelingcells. 25 μL of XTT/PMS solution are added directly to each wellcontaining 100 μL cell culture for 2 hours incubation at 37° C. in a CO₂incubator and absorbance measurements are taking at 450 nm.

Small RNA and miRNA Isolation

Small RNAs including miRNAs are isolated using the miRvana RNA isolationkit (Ambion, Austin, Tex., USA) following the manufacturer's protocol.RNA is quantified using Qubit or Nanodrop spectrophotometer (ThermoFisher, Wilmington, Del., USA) and quality is determined by Agilent 6000nanochip (Agilent Technologies, Palo Alto, Calif., USA).

miRNA Measurement

Quantitative Real-Time PCR Analysis is carried out by as follows: RNAsare reverse transcribed and PCR amplified with miScript reversetranscription kit and miScript SYBR PCR kit (Qiagen, Valencia, Calif.,USA) using ABI 7500 real-time PCR system following the manufacturer'sprotocols. Values from duplicate reactions are averaged and normalizedto the level of U6 SnoRNA. Relative expression levels are calculatedfollowing comparative Ct method as previously described [Schmittgen andLivak. Nat Protoc (2008) 3: 1101-1108]. Alternatively, miRNAs aredetected and relatively quantified using small RNA sequence analysis [asdescribed inwww(dot)illumina(dot)com/techniques/sequencing/rna-sequencing/small-rna-seq(dot)htmlor Wake et al., BMC Genomics (2016) 17(1): 1].

Computational Pipeline to Generate GEiGS Templates

The computational Genome Editing Induced Gene Silencing (GEiGS) pipelineapplies biological metadata and enables an automatic generation of GEiGSDNA templates that are used to minimally edit non-coding RNA genes (e.g.miRNA genes), leading to a new gain of function, i.e. redirection oftheir silencing capacity to target sequence of interest.

As illustrated in FIG. 1, the pipeline starts with filling andsubmitting input: a) target sequence to be silenced by GEiGS; b) thehost organism to be gene edited and to express the GEiGS; c) one canchoose whether the GEiGS would be expressed ubiquitously or not. Ifspecific GEiGS expression is required, one can choose from a few options(expression specific to a certain tissue, developmental stage, stress,heat/cold shock, etc.).

When all the required input is submitted, the computational processbegins with searching among miRNA datasets (e.g. small RNA sequencing,microarray etc.) and filtering (i.e. retaining) only relevant miRNAsthat match the input criteria. Next, the selected mature miRNA sequencesare aligned against the target sequence and miRNA with the highestcomplementary levels are filtered. These naturally target-complementarymature miRNA sequences are then modified to perfectly match the target'ssequence. Then, the modified mature miRNA sequences are run through analgorithm that predicts siRNA potency and the top 20 with the highestsilencing score are filtered. These final modified miRNA genes are thenused to generate 200-500 nt ssDNA or 250-5000 nt dsDNA sequences asfollows:

200-500 nt ssDNA oligos and 250-5000 nt dsDNA fragments are designedbased on the genomic DNA sequence that flanks the modified miRNA. Thepre-miRNA sequence is located in the center of the oligo. The modifiedmiRNA's guide strand (silencing) sequence is 100% complementary to thetarget. However, the sequence of the modified passenger miRNA strand isfurther modified to preserve the original (unmodified) miRNA structure,keeping the same base pairing profile.

Next, differential sgRNAs are designed to specifically target theoriginal unmodified miRNA gene, and not the modified swapping version.Finally, comparative restriction enzyme site analysis is performedbetween the modified and the original miRNA gene and differentialrestriction sites are summarized.

Therefore, the pipeline output includes:

-   -   a) 200-500 nt ssDNA oligo or 250-5000 nt dsDNA fragment sequence        with minimally modified miRNA    -   b) 2-3 differential sgRNAs that target specifically the original        miRNA gene and not the modified    -   c) List of differential restriction enzyme sites among the        modified and original miRNA gene

Selection of GEiGS Precursors:

A list of non-coding RNA types that are both Dicer substrates and areprocessed into small silencing RNA was manually curated from the resultspreviously published in Rybak-Wolf A. et al. [Rybak-Wolf A. et al., Cell(2014) 159, 1153, Äì1167] where the PAR-CLIP technique was used toidentify RNA molecules bound by Dicer and Argonaute 2 and 3. Dicersubstrates were further filtered to exclude regions overlapping withcoding genes, and further curated to remove ambiguous annotations. AGO2and AGO3 smallRNA sequences were processed with cutadapt v1.7 [MartinM., EMBnetjournal (2011) 17(1):10-12] for removing the sequencingadapters. Processed reads where then aligned to GRCh37 assembly of theHuman genome using STAR v2.6.1a [Dobin A. et al., Bioinformatics (2013)29, 15, Äì21] with parameters “-alignIntronMax 1-alignEndsTypeEndToEnd-scoreDelOpen-10000-scoreInsOpen-10000”. Graphics were capturedusing the Integrated Genomics Viewer software [Thorvaldsdóttir H. etal., Brief Bioinform (2013) 14(2):178-92].

Target Genes

miRNAs with ubiquitous expression profile are chosen (depends on theapplication, one might choose miRNAs with expression profile that isspecific to a certain tissue, developmental stage, temperature, stress,etc.).

For example, miRNAs are modified into siRNA targeting the GFP, p53, BAX,PUMA, NOXA genes (see Table 1A, below).

TABLE 1A Target Genes Query sequence Query sequence Gene name IDorganism P53 AB082923 U2OS cells (SEQ ID NO: 7) BAX NM_001291428 U2OScells (SEQ ID NO: 8) eGFP AFA52654 Aequorea victoria (SEQ ID NO: 12)PUMA NM_001127240 (SEQ ID NO: 9) NOXA NM_021127 (SEQ ID NO: 10) FAS1NM_000043 (SEQ ID NO: 11)

siRNA Design

Target-specific siRNAs are designed by publically availablesiRNA-designers such as ThermoFisher Scientific's “BLOCK-iT™ RNAiDesigner” and Invivogen's “Find siRNA sequences”.

sgRNAs Design

sgRNAs are designed to target the endogenous miRNA genes using thepublically available sgRNA designer, as previously described in Park etal. Bioinformatics, (2015) 31(24): 4014-4016. Two sgRNAs are designedfor each cassette, and a single sgRNA is expressed per cell, to initiategene swapping. sgRNAs correspond to the pre-miRNA sequence that ismodified post swapping.

In order to maximize the chance of efficient sgRNA choice, two differentpublicly available algorithms (CRISPER Design:www(dot)crispr(dot)mit(dot)edu:8079/and CHOPCHOP:www(dot)chopchop(dot)cbu(dot)uib(dot)no/) are used and the top scoringsgRNA from each algorithm is selected.

Swapping ssDNA Oligo Design

400 b ssDNA oligo is designed based on the genomic DNA sequence of themiRNA gene. The pre-miRNA sequence is located in the center of theoligo. Next, the double stranded siRNA sequences are swapped with themature miRNA sequences in a way that the guide (silencing) siRNA strandis kept 100% complementary to the target. The sequence of the passengersiRNA strand is modified to preserve the original miRNA structure,keeping the same base pairing profile.

Swapping Plasmid DNA Design

4000 bp dsDNA fragment is designed based on the genomic DNA sequence ofthe miRNA gene. The pre-miRNA sequence is located in the center of thedsDNA fragment. The fragment is cloned into a standard vector (e.g.Bluescript) and transfected into the cells with the Cas9 systemcomponents. Next, the mature miRNA sequences are swapped with the doublestranded siRNA sequences in a way that the guide (silencing) siRNAstrand is kept 100% complementary to the target. The sequence of thepassenger siRNA strand is modified to preserve the original miRNAstructure, keeping the same base pairing profile.

sgRNAs Sequences:

Human miR-150

(SEQ ID NO: 5) 1. CCAGCACTGGTACAAGGGTTGGG (SEQ ID NO: 6)2. CCAACCCTTGTACCAGTGCTGGG

List of Endogenous miRNA that are Swapped:

1. Human miR-150 (SEQ ID NO: 13)

2. Human miR-210 (SEQ ID NO: 14)

3. Human miR-34 (SEQ ID NO: 19-21)

5. Human Let7b (SEQ ID NO: 15)

6. Human miR-184 (SEQ ID NO: 16)

7. Human miR-204 (SEQ ID NO: 17)

8. Human miR-25 (SEQ ID NO: 18)

ssDNA Oligos Used for Gene Swapping:

Oligo-1: GFP-siRNA1_hsa-mir150 (5′→3′) (SEQ ID NO: 1)Oligo-2: GFP-siRNA6_hsa-mir150 (5′→3′) (SEQ ID NO: 2)Oligo-3: TP53-siRNA1_hsa-mir150 (5′→3′) (SEQ ID NO: 3)Oligo-4: TP53-siRNA2_hsa-mir150 (5′→3′) (SEQ ID NO: 4)

Oligo-5: TP53-siRNA1-mMIR17 (5′→3′) (SEQ ID NO: 243) Oligo-6:TP53-siRNA2-mMIR17 (5′3′) (SEQ ID NO: 244) Oligo-7: HPRT-siRNA1-mMIR17(5′→3′) (SEQ ID NO: 245) Oligo-8: HPRT-siRNA2-mMIR17 (5′→3′) (SEQ ID NO:246) Oligo-9: TP53-siRNA1-mMIR21a (5′→3′) (SEQ ID NO: 247) Oligo-10:TP53-siRNA2-mMIR21a (5′→3′) (SEQ ID NO: 248) Oligo11:HPRT-siRNA1-mMIR21a (5′→3′) (SEQ ID NO: 249) Oligo12:HPRT-siRNA2-mMIR21a (5′→3′) (SEQ ID NO: 250) Oligo13: GFP-siRNA1-mMIR17(5′→3′) (SEQ ID NO: 251) Oligo14: GFP-siRNA1-mMIR21a (5′→3′) (SEQ ID NO:252)

sgRNA Cloning

The transfection plasmid utilized is composed of 4 modules comprising of

1) mCherry driven by the CMV promoter terminated by a BGH poly(A) signaltermination sequence;

2) Cas9 (human codon-optimized) driven by the EF1a core promoterterminated by BGH poly(A) signal termination sequence;

3) pol III (U6) promoter sgRNA for guide 1;

Plasmid Design

For transient expression, a plasmid containing three transcriptionalunits is used. The first transcriptional unit contains the EF1a corepromoter-driving expression of Cas9 and the BGH poly(A) signalterminator. The next transcriptional unit consists of CMV promoterdriving expression of mCherry and the BGH poly(A) signal terminator. Thethird contains the pol III (U6) promoter expressing sgRNA to targetmiRNA genes (each vector comprises a single sgRNAs).

Design and Cloning of CRISPR/CAS9 to Target miR-173 and miR-390 andIntroducing SWAPs to Target GFP, AtPDS3 and AtADH1

For proof of concept, the present inventors have designed changes in thesequences of mature miR-173 and miR-390, in their genomic context, totarget GFP, AtPDS3 or AtADH1 (in plant cells), by producing small RNAthat reverse complements target genes. In addition, to maintain thesecondary structure of the miRNA precursor transcript, further changesin the pri-miRNA were carried out (Table 2, below). These fragments werecloned into PUC plasmids and named DONORs and the DNA fragments arereferred as SWAPs. For sequences for modifying miR-173-SWAP1 and SWAP2to target GFP, SWAP3 and SWAP4 to target AtPDS3 and SWAP5 and SWAP10 totarget AtADH1 (see Table 2, below). For sequences for modifyingmiR-390-SWAP5 and SWAP6 to target GFP, SWAP7 and SWAP8 to target AtPDS3and SWAP11 and SWAP12 to target AtADH1 (see Table 2, below).

Guide RNAs targeting miR-173 and miR-390 were introduced intoCRISPR/CAS9 vector system in order to generate a DNA cleavage in thedesired miRNA loci. These were co-introduced to plants with the DONORvectors via gene bombardment protocol, to introduce desiredmodifications through Homologous DNA Repair (HDR). These guide RNAs arespecified in Table 2, below.

TABLE 2 Sequences and oligos used in the experiments SEQ ID NO: Aim 29miR173 30 miR390 31 sgRNA sequence used for miR173 targeting inCRISPR/CAS9 system- GEiGS#4 32 sgRNA sequence used for miR173 targetingin CRISPR/CAS9 system- GEiGS#5 33 sgRNA sequence used for miR390targeting in CRISPR/CAS9 system- GEiGS#1 34 sgRNA sequence used formiR390 targeting in CRISPR/CAS9 system- GEiGS#3 35 mature GEiGS-siRNAtargeting GFP- used in SWAP5 (based on miR390) and in SWAP1 (based onmiR173) 36 Complementary strand of mature GEiGS-siRNA targeting GFP-used in SWAP5 (based on miR390) and in SWAP1 (based on miR173) 37 matureGEiGS-siRNA targeting GFP- used in SWAP6 (based on miR390) and in SWAP2(based on miR173) 38 Complementary strand of mature GEiGS-siRNAtargeting GFP- used in SWAP6 (based on miR390) and in SWAP2 (based onmiR173) 39 mature GEiGS-siRNA targeting AtPDS3- used in SWAP7 (based onmiR390) and in SWAP3 (based on miR173) 40 Complementary strand of matureGEiGS-siRNA targeting AtPDS3- used in SWAP7 (based on miR390) and inSWAP3 (based on miR173) 41 mature GEiGS-siRNA targeting AtPDS3- used inSWAP8 (based on miR390) and in SWAP4 (based on miR173) 42 Complementarystrand of mature GEiGS-siRNA targeting AtPDS3- used in SWAP8 (based onmiR390) and in SWAP4 (based on miR173) 43 mature GEiGS-siRNA targetingAtADH1- used in SWAP11 (based on miR390) and in SWAP9 (based on miR173)44 Complementary strand of mature GEiGS-siRNA targeting AtADH1 - used inSWAP11 (based on miR390) and in SWAP9 (based on miR173) 45 matureGEiGS-siRNA targeting AtADH1- used in SWAP12 (based on miR390) and inSWAP10 (based on miR173) 46 Complementary strand of mature GEiGS-siRNAtargeting AtADH1- used in SWAP12 (based on miR390) and in SWAP10 (basedon miR173) 47 Primary transcript of miR173 (pri-miR173) 48 Primarytranscript of SWAP1 (used in Donor vector for targeting GFP) 49 Primarytranscript of SWAP2 (used in Donor vector for targeting GFP) 50 Primarytranscript of SWAP3 (used in Donor vector for targeting PDS3) 51 Primarytranscript of SWAP4 (used in Donor vector for targeting PDS3) 52 Primarytranscript of SWAP9 (used in Donor vector for targeting ADH1) 53 Primarytranscript of SWAP10 (used in Donor vector for targeting ADH1) 54Primary transcript of miR390 (pri-miR390) 55 Primary transcript of SWAP5(used in Donor vector for targeting GFP) 56 Primary transcript of SWAP6(used in Donor vector for targeting GFP) 57 Primary transcript of SWAP7(used in Donor vector for targeting PDS3) 58 Primary transcript ofSWAP8(used in Donor vector for targeting PDS3) 59 Primary transcript ofSWAP11 (used in Donor vector for targeting ADH1) 60 Primary transcriptof SWAP12 (used in Donor vector for targeting ADH1) 61 Sequence ofmiR173 loci 62 Oligo sequence of SWAP1 (used in Donor vector formodification of miR173 for targeting GFP) 63 Oligo sequence of SWAP2(used in Donor vector for modification of miR173 for targeting GFP) 64Oligo sequence of SWAP3 (used in Donor vector for modification of miR173for targeting PDS3) 65 Oligo sequence of SWAP4 (used in Donor vector formodification of miR173 for targeting PDS3) 66 Oligo sequence of SWAP9(used in Donor vector for modification of miR173 for targeting ADH1) 67Oligo sequence of SWAP10 (used in Donor vector for modification ofmiR173 for targeting ADH1) 68 Oligo sequence of miR390 loci 69 Oligosequence of SWAP5 (used in Donor vector for modification of miR390 fortargeting GFP) 70 Oligo sequence of SWAP6 (used in Donor vector formodification of miR390 for targeting GFP) 71 Oligo sequence of SWAP7(used in Donor vector for modification of miR390 for targeting PDS3) 72Oligo sequence of SWAP8(used in Donor vector for modification of miR390for targeting PDS3) 73 Oligo sequence of SWAP 11 (used in Donor vectorfor modification of miR390 for targeting ADH1) 74 Oligo sequence ofSWAP12 (used in Donor vector for modification of miR390 for targetingADH1) 75 qRT for housekeeping gene- 18S expression (NC_037304 )- Forwardprimer 76 qRT for housekeeping gene- 18S expression (NC_037304 )-Reverse primer 77 qRT for analysis of PDS3 expression (AT4G14210)-Forward primer 78 qRT for analysis of PDS3 expression (AT4G14210)-Reverse primer 79 qRT for analysis of ADH1 expression (AT1G77120)-Forward primer 80 qRT for analysis of ADH1 expression (AT1G77120)-Reverse primer 81 Forward primer for internal amplification of miR390and its modified versions 82 Reverse primer for internal amplificationof miR390 and its modified versions 83 Forward primer for externalamplification of miR390 and its modified versions- primary reaction 84Reverse for external amplification of miR390 and its modified versions-primary reaction 85 Forward primer for external amplification of miR390and its modified versions- nested reaction 86 Reverse for externalamplification of miR390 and its modified versions- nested reaction 87Forward primer for internal amplification of miR173 and its modifiedversions 88 Reverse primer for internal amplification of miR173 and itsmodified versions 89 Forward primer for external amplification of miR173and its modified versions- primary reaction 90 Reverse for externalamplification of miR173 and its modified versions- primary reaction 91Forward primer for external amplification of miR173 and its modifiedversions- nested reaction 92 Reverse for external amplification ofmiR173 and its modified versions- nested reaction

Plasmid Transfection

For transfection Lipofectamine® 2000 Transfection Reagent (or any other)is used according to the manufacturer's protocol, in short:

For adherent cells: One day before transfection, 0.5-2×10⁵ cells areplated in 500 μl of growth medium without antibiotics so that cells willbe 90-95% confluent at the time of transfection.

For suspension cells: Just prior to preparing complexes, 4-8×10⁵ cellsin 500 μl of growth medium are plated without antibiotics.

For each transfection sample, complexes are prepared as follows: a) DNAis diluted in 50 μl of Opti-MEM® I Reduced Serum Medium without serum(or other medium without serum) and is mixed gently. b) Lipofectamine™2000 is mixed gently before use, then the appropriate amount is dilutedin 50 μl of Opti-MEM® I Medium, and is incubated for 5 minutes at roomtemperature. It should be noted that proceeding into step c should beeffected within 25 minutes. c) After the 5 minute incubation, thediluted DNA is combined with diluted Lipofectamine™ 2000 (totalvolume=100 μl) is mixed gently and incubated for 20 minutes at roomtemperature (solution may appear cloudy). It should be noted that thecomplexes are stable for 6 hours at room temperature. d) 100 μl of thecomplexes is added to each well containing cells and medium and is mixedgently by rocking the plate back and forth. e) cells are incubated at37° C. in a CO₂ incubator for 18-48 hours prior to testing for transgeneexpression. Medium may be changed after 4-6 hours.

FACS Sorting of Fluorescent Protein-Expressing Cells

48 hrs after plasmid/RNA delivery, cells are collected and sorted forfluorescent protein expression (e.g. mCherry) using a flow cytometer inorder to enrich for fluorescent protein/editing agent expressing cellsas previously described [Chiang et al., Sci Rep (2016) 6: 24356]. Thisenrichment step allows bypassing antibiotic selection and collection ofonly cells transiently expressing the fluorescent protein, Cas9 and thesgRNA. These cells can be further tested for editing of the target geneby HR events followed by efficient silencing of the target gene i.e.GFP.

Bombardment and Plant Regeneration

Arabidopsis Root Preparation:

Chlorine gas sterilised Arabidopsis (cv. Col-0) seeds were sown on MSminus sucrose plates and vernalised for three days in the dark at 4° C.,followed by germination vertically at 25° C. in constant light. Aftertwo weeks, roots were excised into 1 cm root segments and placed onCallus Induction Media (CIM: 1/2 MS with B5 vitamins, 2% glucose, pH5.7, 0.8% agar, 2 mg/l IAA, 0.5 mg/l 2,4-D, 0.05 mg/l kinetin) plates.Following six days incubation in the dark, at 25° C., the root segmentswere transferred onto filter paper discs and placed onto CIMM plates,(1/2 MS without vitamins, 2% glucose, 0.4 M mannitol, pH 5.7 and 0.8%agar) for 4-6 hours, in preparation for bombardment.

Bombardment

Plasmid constructs were introduced into the root tissue via thePDS-1000/He Particle Delivery (Bio-Rad; PDS-1000/He System #1652257),several preparative steps, outlined below, were required for thisprocedure to be carried out.

Gold Stock Preparation

40 mg of 0.6 μm gold (Bio-Rad; Cat: 1652262) was mixed with 1 ml of 100%ethanol, pulse centrifuged to pellet and the ethanol removed. This washprocedure was repeated another two times.

Once washed the pellet was resuspended in 1 ml of sterile distilledwater and dispensed into 1.5 ml tubes of 50 μl aliquot working volumes.

Bead Preparation

In short, the following was performed:

A single tube was sufficient gold to bombard 2 plates of Arabidopsisroots, (2 shots per plate), therefore each tube was distributed between4 1,100 psi Biolistic Rupture disks (Bio-Rad; Cat: 1652329).

Bombardments requiring multiple plates of the same sample, tubes werecombined and volumes of DNA and CaCl2/spermidine mixture adjustedaccordingly, in order to maintain sample consistency and minimiseoverall preparations.

The following protocol summarises the process of preparing one tube ofgold, these should be adjusted according to number of tubes of goldused.

All subsequent processes were carried out at 4° C. in an Eppendorfthermomixer.

Plasmid DNA samples were prepared, each tube comprising 11 μg of DNAadded at a concentration of 1000 ng/μl

1) 493 μl ddH2O was added to 1 aliquot (7 μl) of spermidine(Sigma-Aldrich; S0266), giving a final concentration of 0.1 Mspermidine. 1250 μl 2.5M CaCl2 was added to the spermidine mixture,vortexed and placed on ice.

2) A tube of pre-prepared gold was placed into the thermomixer, androtated at a speed of 1400 rpm.

3) 11 μl of DNA was added to the tube, vortexed, and placed back intothe rotating thermomixer.

4) To bind, DNA/gold particles, 70 μl of spermidine CaCl2 mixture wasadded to each tube (in the thermomixer).

5) The tubes were vigorously vortexed for 15-30 seconds and placed onice for about 70-80 seconds.

6) The mixture was centrifuged for 1 minute at 7000 rpm, the supernatantwas removed and placed on ice.

7) 500 μl 100% ethanol was added to each tube and the pellet wasresuspended by pipetting and vortexed.

8) The tubes were centrifuged at 7000 rpm for 1 minute.

9) The supernatant was removed and the pellet resuspended in 50 μl 100%ethanol, and stored on ice.

Macro Carrier Preparation

The following was performed in a laminar flow cabinet:

1) Macro carriers (Bio-Rad; 1652335), stopping screens (Bio-Rad;1652336), and macro carrier disk holders were sterilised and dried.

2) Macro carriers were placed flatly into the macro carrier diskholders.

3) DNA coated gold mixture was vortexed and spread (5 μl) onto thecentre of each Biolistic Rupture disk.

Ethanol was allowed to evaporate.

PDS-1000 (Helium Particle Delivery System)

In short, the following was performed:

The regulator valve of the helium bottle was adjusted to at least 1300psi incoming pressure. Vacuum was created by pressing vac/vent/holdswitch and holding the fire switch for 3 seconds. This ensured heliumwas bled into the pipework.

1100 psi rupture disks were placed into isopropanol and mixed to removestatic.

1) One rupture disk was placed into the disk retaining cap.

2) Microcarrier launch assembly was constructed (with a stopping screenand a gold containing microcarrier).

3) Petri dish Arabidopsis root callus was placed 6 cm below the launchassembly.

4) Vacuum pressure was set to 27 inches of Hg (mercury) and helium valvewas opened (at approximately 1100 psi).

5) Vacuum was released; microcarrier launch assembly and the rupturedisk retaining cap were removed.

6) Bombardment on the same tissue (i.e. each plate was bombarded 2times).

7) Bombarded roots were subsequently placed on CIM plates, in the dark,at 25° C., for additional 24 hours.

Co-Bombardments

When bombarding GEiGS plasmids combinations, 5 μg (1000 ng/μl) of thesgRNA plasmid was mixed with 8.5 μg (1000 ng/μl) swap plasmid and 11 μlof this mixture was added to the sample. If bombarding with more GEiGSplasmids at the same time, the concentration ratio of sgRNA plasmids toswap plasmids used was 1:1.7 and 11 μg (1000 ng/μl) of this mixture wasadded to the sample. If co-bombarding with plasmids not associated withGEiGS swapping, equal ratios were mixed and 11 μg (1000 ng/μl) of themixture was added to each sample.

Plant Regeneration

For shoot regeneration, modified protocol from Valvekens et al.[Valvekens, D. et al., Proc Natl Acad Sci USA (1988) 85(15): 5536-5540]was carried out. Bombarded roots were placed on Shoot Induction Media(SIM) plates, which included 1/2 MS with B5 vitamins, 2% glucose, pH5.7, 0.8% agar, 5 mg/l 2 iP, 0.15 mg/l IAA. Plates were left in 16 hourslight at 25° C.-8 hours dark at 23° C. cycles. After 10 days, plateswere transferred to MS plates with 3% sucrose, 0.8% agar for a week,then transferred to fresh similar plates. Once plants regenerated, theywere excised from the roots and placed on MS plates with 3% sucrose,0.8% agar, until analysed.

Phenotypic Analysis

As described above, such as by looking at the fluorescence and cellmorphology or other phenotypes such as growth rate/inhibition and/orapoptosis that are dependent on the target gene such Nutlin3 resistancein the case of TP53 silencing.

Anti-Viral Assay

The assay is based on cytopathic effect (CPE) commonly used to determinethe potency of purified interferon stocks. In the CPE assay, anti-viralactivity is measured based on its ability to inhibit virus-inducedcytopathology as measured by a crystal violet live-cell stain[previously described by Rubinstein et al., J Virol. (1981) 10:755-758].

VSV forms discrete, microscopic plaques in stationary cultures of theWISH amnion cell line. Microplaque formation is rapid, reproducible, andeasily quantitated, occurs at temperatures ranging from 33 to 40° C.,and does not require a semisolid overlay.

Allyl Alcohol Selection

For selection of plants with allyl alcohol, 10 days post bombardment,roots were placed on SIM media. Roots were immersed in 30 mM allylalcohol (Sigma-Aldrich, US) for 2 hours. Then the roots were washedthree times with MS media, and placed on MS plates with 3% sucrose, 0.8%agar. Regeneration process was carried on as previously described.

Genotyping

Plant tissue samples were treated and amplicons amplified in accordanceto the manufacturers recommendations. MyTaq Plant-PCR Kit (BioLine BIO25056) for short internal amplification and Phire Plant Direct PCR Kit(Thermo Scientific; F-130WH) for longer external amplifications. Oligosused for these amplifications are specified in Table 2, above. Differentmodifications in the miRNA loci were identified through differentdigestion patterns of the amplicons, as follows:

For modifications of miR-390-internal amplicon was 978 base pairs long,and for external amplification it was 2629 base pairs. For theidentification of swap 7, digestion with NlaIII resulted in a fragmentsize of 636 base pairs, while in the wt version it was cleaved to 420and 216 long fragments. For the identification of swap 8, digestion withHpy188I resulted in fragments size of 293 and 339 base pairs, while inthe wt version this site was absent and resulted in a 632-long fragment.For the identification of swaps 11 and 12, digestion with Bccl resultedin a fragment size of 662 base pairs, while in the wt version it wascleaved to 147 and 417 long fragments.

For modifications of miR-173—internal amplicon was 574 base pairs long,and for nested external amplification it was 466 base pairs. For theidentification of swap 3, digestion with BsII resulted in fragments sizeof 217 and 249 base pairs in the external amplicon and 317 and 149 inthe internal one. In the wt version this site was absent and resulted ina 466-long fragment in the external amplicon and 574 in the internalreaction. For the identification of swap 4, digestion with BtsαIresulted in fragments size of 212 and 254 base pairs in the externalamplicon and 212 and 362 in the internal one. In the wt version, thissite was absent and resulted in a 466-long fragment in the externalamplicon and 574 in the internal reaction. For the identification ofswap 9, digestion with NlaIII resulted in fragments size of 317 and 149base pairs in the external amplicon and 317 and 244 in the internal one.In the wt version, this site was absent and resulted in a 466-longfragment in the external amplicon and 561 in the internal reaction. Forthe identification of swap 10, digestion with NlaIII resulted infragments size of 375 and 91 base pairs in the external amplicon and 375and 186 in the internal one. In the wt version, this site was absent andresulted in a 466-long fragment in the external amplicon and 561 in theinternal reaction.

DNA and RNA Isolation

Plant samples were harvested into liquid nitrogen and stored in −80° C.until processed. Grinding of tissue was carried out in tubes placed indry ice, using plastic Tissue Grinder Pestles (Axygen, US). Isolation ofDNA and total RNA from ground tissue was carried out using RNA/DNAPurification kit (cat. 48700; Norgen Biotek Corp., Canada), according tomanufacturer's instructions. In the case of low 260/230 ratio (<1.6), ofthe RNA fraction, isolated RNA was precipitated overnight in −20° C.,with 1 μl glycogen (cat. 10814010; Invitrogen, US) 10% V/V sodiumacetate, 3 M pH 5.5 (cat. AM9740, Invitrogen, US) and 3 times the volumeof ethanol. The solution was centrifuged for 30 minutes in maximumspeed, at 4° C. This was followed by two washes with 70% ethanol,air-drying for 15 minutes and resuspending in Nuclease-free water (cat.10977035; Invitrogen, US).

Reverse Transcription (RT) and Quantitative Real-Time PCR (qRT-PCR)

One microgram of isolated total RNA was treated with DNase I accordingto manufacturer's manual (AMPD1; Sigma-Aldrich, US). The sample wasreverse transcribed, following the instructor's manual of High-CapacitycDNA Reverse Transcription Kit (cat 4368814; Applied Biosystems, US).

For gene expression, Quantitative Real Time PCR (qRT-PCR) analysis wascarried out on CFX96 Touch™ Real-Time PCR Detection System (BioRad, US)and SYBR® Green JumpStart™ Taq ReadyMix™ (S4438, Sigma-Aldrich, US),according to manufacturers' protocols, and analysed with Bio-RadCFXmanager program (version 3.1). For the analysis of AtADH1 (AT1G77120)the following primer set was used: Forward GTTGAGAGTGTTGGAGAAGGAG SEQ IDNO: 237 and reverse CTCGGTGTTGATCCTGAGAAG SEQ ID NO: 238; For theanalysis of AtPDS3 (AT4G14210), the following primer set was used:Forward GTACTGCTGGTCCTTTGCAG SEQ ID NO: 239 and reverseAGGAGCACTACGGAAGGATG SEQ ID NO: 240; For endogenous calibration gene,the 18S ribosomal RNA gene (NC_037304) was used—ForwardACACCCTGGGAATTGGTTT SEQ ID NO: 241 and reverse GTATGCGCCAATAAGACCAC SEQID NO: 242.

Example 1A Genome Editing Induced Gene Silencing (GEiGS) Platform

MicroRNAs (miRNAs) MicroRNAs (miRNAs) are small endogenous non-codingRNAs (ncRNAs) of 20 to 24-nucleotide in length, originating from longself-complementary precursors. Mature miRNAs regulate gene expression intwo ways; (i) by inhibiting translation or (ii) by degrading codingmRNAs by perfect or near-perfect complement with the target mRNAs. Inanimals, seminal studies on miRNAs have shown that only the seed region(sequence spanning from position 2 to 8 at the 5′ end), is crucial fortarget recognition. The seed sequence pairs fully to its responsiveelement mainly at the 30-untranslated region (UTR) of the target mRNA.The alteration of miRNAs biogenesis mechanism, miRNAs expression leveland miRNAs regulatory networks affects important biological pathwayssuch as cellular differentiation and apoptosis and it is detected invarious human diseases and syndromes, especially in cancer.

All tumors present specific signatures of miRNAs altered expression. Forthis reason, miRNAs expression profiles of tumors may represent validand useful biomarkers for diagnosis, prognosis, patient stratification,definition of risk groups and monitoring of the response to therapy.Equally relevant is the emerging role of miRNAs in viral infections.Data from literature show a mutual interference between viruses and thehost cell's miRNA machinery. For instance, viruses may impair the hostcell's miRNA pathway by interacting with specific proteins, synthesizetheir own miRNAs to modify cellular environment or to regulate their ownmRNAs, or make use of cellular miRNAs to their favor. However, it isalso true that host cell's miRNAs may target viral mRNAs. In many cases,this bidirectional interference is resolved in favor of the viruses thatas a result may escape the immune response and complete the replicationcycle.

Accordingly, the present inventors are utilizing endogenous ncRNAsequences (e.g. of miRNA) that are re-designed using GEiGS to gainsilencing functionality, by Homologous Recombination (HR), in order tospecifically silent any RNA of interest. In order to replace chosensequences, HR uses longer stretches of sequence homology flanking theDSB site to repair DNA lesions and is therefore considered to beaccurate mechanism for DSB repair due to the requirement of highersequence homology between the damaged and intact donor strands of DNA(i.e. the inserted siRNA sequence). This process is considered to beerror-free if the DNA template used for repair is identical to theoriginal DNA sequence at the DSB, or it can introduce very specificmutations into the damaged DNA e.g. swapping genes.

Example 1B Genome Editing Induced Gene Silencing (GEiGS)

In order to design GEiGS oligos, template non-coding RNA molecules(precursors) that are processed and give raise to derivate smallsilencing RNA molecules (matures) are required. The present inventorshave characterized dicer substrate RNAs (i.e. cellular RNAs that arebound by Dicer) that produce silencing engaged small RNAs (i.e. smallRNAs that are bound by Argonaut 2 and Argonaut 3) in human and C.elegans as previously discussed in Rybak-Wolf [Rybak-Wolf, A. et al.,Cell (2014) 159: 1153, Äì1167]. Crossing both datasets (dicer bound RNAs& Ago2 and Ago3 bound small RNAs), allowed to generate a list ofnon-coding RNAs that are precursors of small silencing RNAs (FIG. 10 andFIGS. 11A-E). Two sources of precursor and their corresponding maturesequences were used for generating GEiGS oligos. For miRNAs, sequenceswere obtained from the miRBase database [Kozomara, A. andGriffiths-Jones, S., Nucleic Acids Res (2014) 42: D68,AID73]. Other typeof precursors (including tRNAs, snRNAs, and various types of repeats)were obtained from a recent publication describing Dicer-bound &AGO-bound RNAs [Rybak-Wolf, A. et al., Cell (2014) 159: 1153, Äì1167].

Silencing targets were chosen in a variety of host organisms. siRNAswere designed against these targets using the siRNArules software[Holen, T., RNA (2006) 12: 1620, Äì1625.]. Each of these siRNA moleculeswas used to replace the mature sequences present in each precursor,generating “naive” GEiGS oligos. The structure of these naive sequenceswas adjusted to approach the structure of the wild type precursor asmuch as possible using the ViennaRNA Package v2.6 [Lorenz, R. et al.,ViennaRNA Package 2.0. Algorithms for Molecular Biology (2011) 6: 26].Examples for successful structure maintenance versus non-successfulstructure maintenance can be found in FIG. 12A-D. After the structureadjustment, the number of sequence and secondary structure changesbetween the wild type and the modified oligo were calculated. Thesecalculations are essential to identify potentially functional GEiGSoligos that require minimal sequence changes with respect to the wildtype (FIG. 12A-E).

CRISPR/cas9 small guide RNAs (sgRNAs) against the wild type precursorswere generated using the CasOT software [Xiao, A. et al., Bioinformatics(2014) 30: 1180, Äì1182]. sgRNAs were selected where the modificationsapplied to generate the GEiGS oligo affect the PAM region of the sgRNA,rendering it ineffective against the modified oligo.

Example 2 GEiGS of an “Endogenous” Transgene

A quick and robust approach to check the efficiency of GEiGS is tosilence a transgene, which will serve as endogenous gene and in additionis also a marker gene like GFP (green fluorescent protein). There arefew options to assess the effectiveness of GFP silencing in cells, thepresent inventors are using FACS analysis, RT-qPCR and microscopy toassess the effectiveness of GFP silencing in cells.

Silencing of GFP is well characterized and there are many availableshort interfering RNA sequences (siRNA) that are efficient in triggeringGFP silencing. Therefore, for gene swapping, the present inventors areusing 21 mer siRNA molecules designed to silence GFP. Additionally oralternatively, the present inventors are using public algorithms thatpredict which siRNA will be effective in initiating gene silencing to agiven gene (e.g. GFP). Since the predictions of these algorithms are not100%, the present inventors are only using sequences that are theoutcome of at least two different algorithms.

In order to use siRNA sequences that will silence the GFP gene, thepresent inventors are swapping them with a known endogenous miRNA genesequences using the CRISPR/Cas9 system. There are many databases ofcharacterized miRNAs, the present inventors are choosing several knownhuman miRNAs with different expression profiles (e.g. low constitutiveexpression, highly expressed, induced in stress, etc.). In order to swapthe endogenous miRNA sequence with siRNA the present inventors are usingthe HR approach.

As illustrated in FIG. 2, using HR the present inventors arecontemplating two options: 1) use a donor ssDNA oligo sequence of around200-500 bases which includes the swapping siRNA sequence in the middleor 2) use plasmids expressing 1 Kb-4 Kb insert which is almost 100%identical to the miRNA surrounding in the genome except the 2×21 bp ofthe miRNA and the *miRNA that is changed into the siRNA of the GFP(500-2000 bp up and downstream the siRNA). The transfection includes afew constructs: CRISPR:Cas9/RFP sensor to track and enriched forpositive transformed cells, gRNAs that guide the Cas9 to produce a DSBwhich is repaired by HR depending on the insertion vector/oligo.

The insertion vector contains two continuous regions of homologysurrounding the targeted locus that are replaced (e.g. miRNA) and ismodified to carry the mutation of interest (i.e. siRNA). If plasmid isused, the targeting construct is used as a template for homologousrecombination ending with the replacement of the miRNA with the siRNA ofchoice. After transfection to tissue culture cells, FACS is used toenrich for positive Cas9/sgRNA transfected events, cells are scored forGFP silencing under microscope (as illustrated in FIG. 2). It isexpected that the positive edited cells will produce siRNA sequencestargeting the GFP gene and therefore the GFP expression of the transgenewill be silenced compared to control cells.

In order to show proof of concept (POC) of GFP silencing using GEiGS,transgenic human cell lines including U20S, RPE1, A549 or Hela cellsthat express GFP are being utilized. Cells are transfected with GEiGSmethodology and with cassettes to swap endogenous non-coding RNAs (e.g.miRNA) and turn it into a non-coding RNA that is processed into siRNAstargeting GFP to initiate the RNA silencing mechanism against GFP. Asillustrated in FIGS. 3A-B, knock down of GFP gene expression levels inhuman cells results in reduced expression of GFP in cells expressingsiGFP (i.e. in which GFP is silenced) as compared to control cells (FIG.3A).

Example 3 GEiGS of Exogenous Transgene (GFP) in Tissue Culture Cells

In addition to the former example of GFP silencing (Example 2 above),another way to demonstrate the efficiency of GEiGS is to silence amarker gene like GFP in a transient GFP transfection assay. Asillustrated in FIG. 4, human cells are treated using GEiGS in order toredirect silencing specificity of endogenous miRNA through expression ofsmall siRNA molecules targeting the GFP gene (as discussed in Example 2,above). Control untreated cells and GEiGS-GFP cells (i.e. expressingsiGFP) are then transfected with a plasmid expressing separately twomarkers (sensor) GFP+RFP (Red Fluorescent Protein), cells which expressonly RFP but not GFP in the GEiGS treatment are the results of GFP genesilencing due to siGFP expression. DNA from these cells (Red but lack ofGFP expression) are extracted and examined for the correctgenome-editing event. Furthermore, the cells can be analyzed for theloss of expression of GFP e.g., by fluorescent detection of GFP orq-PCR, HPLC.

Example 4 GEiGS of TP53 or HPRT Expression Inhibits Nutlin3-Induced or6TG (Thioguanine, 6-TG, 6-Tioguanine) Cell Death/Growth Inhibition inU2OS and RPE1 or Mouse Embryonic Stem (mES) Cells

To show POC of GEiGS in human cells, the present inventors are workingwith U2OS, RPE1 or mouse embryonic stem cells. U2OS are cells that growfast and are easy to transfect with high efficiency. These cellsoriginate from bone cancer—osteosarcoma. RPE1 are epithelial cellsoriginated from normal retina (i.e. not from a disease or sick culture)with normal and active TP53 as do mES.

TP53 is a tumor-suppressor protein that induces directly or indirectlyapoptotic cell death in response to oncogenic stress. The consequencesof DNA damage depend on the cell type and the severity of the damage.Mild DNA damage can be repaired with or without cell-cycle arrest. Moresevere and irreparable DNA injury leads to the appearance of cells thatcarry mutations or causes a shift towards induction of the senescence orcell death programs. Although for many years it was argued that DNAdamage kills cells via apoptosis or necrosis, technical andmethodological progress during the last few years has helped to revealthat this injury might also activate death by autophagy or mitoticcatastrophe, which may then be followed by apoptosis or necrosis. Themolecular basis underlying the decision-making process is currently thesubject of intense investigation.

Today, anyone with interest in cancer research is already well aware ofthe existence of TP53 and its relevance to practically every aspect oftumor biology. TP53 is undoubtedly one of the most extensively studiedgenes and proteins. Early studies indicate thattransactivation-defective mutants of p53 are capable of inducingapoptosis, implying a transcription-independent role for p53 inapoptosis. DNA-damage leads to mitochondrial translocation of TP53. TP53binds to Bcl-2 family protein Bcl-xL to influence cytochrome c release.TP53 directly activates the proapoptotic Bcl-2 protein Bax in theabsence of other proteins to permeabilize mitochondria and engage theapoptotic program. TP53 can release both proapoptotic multidomainproteins and BH3-only proteins that are sequestered by Bcl-Xl. Inaddition, TP53 can directly mediate mitochondrial mechanism of apoptosisby facilitating Bax oligomerization, binding to Bcl-xL, but not to Bax,TP53-Bcl-xL interaction releases Bax and released Bax forms oligomers inmitochondrial membrane, leading cytochrome c release and apoptosis (theproline-rich domain, aa 62-91 in mouse, of TP53 is required for thiseffect) [Jerry et al. Science (2004) 303(5660):1010-4]. TP53 also act asa transcription factor promoting the expression of the pro-apoptoticgenes such as BAX, PUMA and NOXA.

As illustrated in FIG. 5, the present inventors are modifying RPE1 cellsto express siRNA directed against TP53, these cells when exposed toNutlin3 or chemotherapy (e.g. Camptothecin (CPT), etoposide, olaparib,etc.) show inhibition of cell death. One of the assays the presentinventors are utilizing is the crystal violet assay in which staining ofcells enable to compare cell number (density) and morphology, whichdiffer between healthy and dying cells. Cell clones that are resistantto cell death are verified to the correct genome editing event and forexpression of the relevant TP53 siRNA. Furthermore, the cells can beanalyzed for the loss of expression of TP53 e.g., by fluorescentdetection of GFP or q-PCR, HPLC.

Tioguanine, also known as thioguanine or 6-thioguanine (6-TG) is amedication commonly used to treat acute myeloid leukemia (AML), acutelymphocytic leukemia (ALL), and chronic myeloid leukemia (CML).Tioguanine, an antimetabolite, is a purine analogue of guanine and worksby disrupting DNA and RNA. 6-Thioguanine is a thio analogue of thenaturally occurring purine base guanine. 6-thioguanine utilises theenzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRTase/HPRT) tobe converted to 6-thioguanosine monophosphate (TGMP). Highconcentrations of TGMP may accumulate intracellularly and hamper thesynthesis of guanine nucleotides via the enzyme Inosine monophosphatedehydrogenase (IMP dehydrogenase). TGMP is converted by phosphorylationto thioguanosine diphosphate (TGDP) and thioguanosine triphosphate(TGTP). Simultaneously deoxyribosyl analogs are formed, via the enzymeribonucleotide reductase. The TGMP, TGDP and TGTP are collectively named6-thioguanine nucleotides (6-TGN). 6-TGN are cytotoxic to cells by: (1)incorporation into DNA during the synthesis phase (S-phase) of the cell;and (2) through inhibition of the GTP-binding protein (G protein) Rac1,which regulates the Rac/Vav pathway. An additional effect may be derivedfrom the incorporation of 6-thioguanine into RNA. This yields a modifiedRNA strand which cannot be read by the ribosomes.

In brief, loss or reduction of HPRT gene expression render the cellsresistant to 6TG. Accordingly, the present inventors are modifying HPRTgene expression by expressing siRNA directed against HPRT, and analyzingdownregulation of HPRT by resistance to 6TG.

Example 5 GEiGS of Pro-Apoptotic Genes (BAX, PUMA, NOXA) InhibitsChemotherapy-Induced Cell Death in Human Cancer Cells

In this experiment the present inventors are using U2OS cells. In orderto create cells resistant to chemotherapy agents like CPT, etoposide,olaparib, etc., the present inventors are first using siRNA capable oftargeting apoptotic genes like BAX, PUMA and NOXA which are known aspro-apoptotic genes.

As illustrated in FIG. 6, the present inventors are treating U2OS cellsusing GEiGS to express siRNA targeting apoptotic genes. Modified cellsthat express siRNA are expected to be resistant to chemotherapy (e.g.like CPT, etoposide, olaparib, etc.)-induced cell death. Aftertransfection with GEiGS cassettes+RFP sensor, transfected cells areenriched with FACS and cells are exposed to chemotherapy agents. In thecontrol, all cells are sensitive and die or enter senescence (easy todetect under a microscope using Dapi staining, few cells with bignuclei). Clones that are resistant to cell death and or senescence areexpected to be positively expressing edited siRNAs and are verified tothe have the correct genome editing modification and expression of therelevant siRNA. Furthermore, the cells can be analyzed for the loss ofexpression of apoptotic genes like BAX, PUMA and NOXA e.g., byfluorescent detection of GFP or q-PCR, HPLC.

Example 6 Utilizing GEiGS to Immunize Human Cells Against ViralInfection

In order to prove that GEiGS is a robust method for human immunizationwith the ability to knock down exogenous pathogenic gene, the presentinventors are providing an example of silencing of a virus gene. Alentiviral system is very effective at delivering genetic material towhole model organisms and almost all mammalian cells, includingnon-dividing non growing cells, as well as difficult-to-transfect cellsincluding neuron, primary and stem cells. The efficiency of lentiviraltransduction is close to 100% depending on the Multiplicity Of Infection(MOI), making it ideal as an expression vector system.

Control cells that are infected with lentivirus expressing-GFP showexpression of GFP under the microscope (as illustrated in FIG. 7).GEiGS-GFP cells engineered to express siRNA targeting GFP gene (asillustrated in Example 2, above) are expected to show reduced levels ofGFP (as illustrated in FIG. 7). Generating GEiGS cells with no or lowGFP gene expression after infection with Virus-GFP (e.g. Lenti-GFP) willprove that silencing of exogenous gene was achieved and that GEiGS is aneffective method to immunize human cells against invasive infectious RNAlike viruses.

There are few easy options to assess the effectiveness of the GFP genesilencing in the cell, the present inventors are using FACS analysis,RT-qPCR, microscopy and/or immunoblotting. Therefore, for gene swapping,the present inventors designed 21 mer siRNA molecules (as described inExample 2, above). The present inventors are using public algorithmsthat predict which siRNA will be effective in initiating gene silencingto a given gene (as described in Example 2, above).

Example 7 Immunizing Human Cells to Virus Infection by Silencing of anExogenous Virus Gene (Cell Survival Assay)

In order to prove that GEiGS is a robust method for human immunizationwith the ability to knock down exogenous genes, in addition to exampleusing lentivirus expressing GFP (Example 6, above), the presentinventors are using wild-type RNA virus infection and are scoring forcell survival. The present inventors are providing an example ofsilencing of a Vesicular stomatitis virus (VSV) gene.

VSV, a Rhabdoviridae RNA virus, can infect many cell types and thereforeis a common laboratory virus used to study the properties of viruses inthe family Rhabdoviridae, as well as to study viral evolution. VSV is anarbovirus, and its replication occurs in the cytoplasm. The genome ofVSV is on a single molecule of negative-sense RNA that has 11,161nucleotides in length that encodes five major proteins: G protein (G),large protein (L), phosphoprotein, matrix protein (M) and nucleoprotein.In healthy human cells, the virus cannot reproduce (probably because ofthe interferon response) but in many cancer cells (that have a reducedinterferon response) VSV can grow and hence lyse the oncogenic cells. Afunctional anti-viral assay based on cytopathic effect (CPE) is utilizedto determine cell survival as described in detail in the ‘generalmaterials and experimental procedures’ section above. This method allowsevaluating and comparing cell survival and viability. Through stainingcells it is possible to compare cell number, density and morphology,which differ between healthy and dying cells.

In order to find efficient siRNAs targeting VSV genes, a preliminaryexperiment with different transfection of siRNAs targeting virus genesis carried out. siRNAs that inhibit VSV-induced cell death are used withGEiGS to edit human WISH cells to express these siRNAs. Control cellsthat are infected with VSV will show cytopathology effect as measured bya crystal violet compared to GEiGS cells that are expected to beresistant to virus infection.

Example 8 GEiGS of the Pro-Apoptotic FAS Gene Expression Reduces5-Fluorouracil-Induced Apoptosis in HCT116 Cells

It was previously shown by Pedro et al. [Pedro et al. Biochimica etBiophysica Acta (2007) 1772: 40-47] that in HCT116 human colorectalcancer cells expressing wild-type p53, silencing of FAS expression byRNA interference moderates 5-FU-induced apoptosis.

HCT116 cells are treated using GEiGS to express siRNA targeting FASgene. HCT116 control and GEiGS positive cells (expressing FAS siRNA) aretreated with 5-FU (e.g. 1-8 μM) for e.g. 8-48 hours. Cell viability isevaluated by XTT and trypan blue dye exclusion. Apoptosis is assessed bychanges in nuclear morphology and caspase 3 activity. 5-FU is cytotoxicin HCT116 cells but when siRNA is used to inhibit Fas, 5-FU-mediatednuclear fragmentation and caspase 3 activity are expected to be markedlyreduced.

Example 9 Generation of Plants with Modified Endogenous miRNA to TargetDifferent Genes

Minimal modifications in the genomic loci of a miRNA, in its recognitionsequence (which will mature to a miRNA) can lead to a new system toregulate new genes, in a non-transgenic manner. Therefore, anagrobacterium-free transient expression method was used, to introducethese modifications by bombardment of Arabidopsis roots, and theirregeneration for further analysis. The present inventors had chosen totarget two genes, PDS3 and ADH1 in Arabidopsis plants.

Carotenoids play an important role in many physiological processes inplants and the phytoene desaturase gene (PDS3) encodes one of theimportant enzymes in the carotenoid biosynthesis pathway, its silencingproduces an albino/bleached phenotype. Accordingly, plants with reducedexpression of PDS3 exhibit reduced chlorophyll levels, up to completealbino and dwarfism.

Alcohol dehydrogenase (ADH1) comprises a group of dehydrogenase enzymeswhich catalyse the interconversion between alcohols and aldehydes orketones with the concomitant reduction of NAD+ or NADP+. The principalmetabolic purpose for this enzyme is the breakdown of alcoholic toxicsubstances within tissues. Plants harbouring reduced ADH1 expressionexhibit increase tolerance to allyl alcohol. Accordingly, plants withreduced ADH1 are resistant to the toxic effect of allyl alcohol,therefore their regeneration was carried out with allyl alcoholselection. Two well-established miRNAs were chosen to be modified,miR-173 and miR-390, that were previously shown to be expressedthroughout plant development [Zielezinski A et al., BMC Plant Biology(2015) 15: 144]. To introduce the modification, a 2-component system wasused. First, the CRISPR/CAS9 system was used, to generate a cleavage inthe miR-173 and miR-390 loci, through designed specific guide RNAs(Table 2, above), to promote homologous DNA repair (HDR) in the site.Second, A DONOR sequence, with the desired modification of the miRNAsequence, to target the newly assigned genes, was introduced as atemplate for the HDR (Table 2, above). In addition, since the secondarystructure of the primary transcript of the miRNA (pri-miRNA) isimportant for the correct biogenesis and activity of the mature miRNA,further modifications were introduced in the complementary strand in thepri-miRNA and analysed in mFOLD(www(dot)unafold(dot)rna(dot)Albany(dot)edu) for structure conservation(data not shown). In total, two guides were designed for each miRNAloci, and two different DONOR sequences (modified miRNA sequences) weredesigned for each gene (Table 2, above).

Example 10 Bombardment and Plant Regeneration

GEiGS constructs were bombarded into pre-prepared roots (as discussed indetail in the materials and experimental procedures section, above) andregenerated. Plantlets were selected via bleached phenotype for PDS3transformants and survival on allyl alcohol treatment for ADH1transformants. In order to validate Swap compared to no Swap, i.e.retained wild type, these plants were subsequently screened forinsertion through specific primers spanning the modified region followedby restriction digest (FIG. 13).

Example 11 Genotype Validation of Phenotype Selection

As discussed above, the Proof of Concept (POC) for the gene editingsystem was established using well known phenotypic traits, Phytoenedesaturase (PDS3) and Alcohol desaturase (ADH1) as targets.

As mentioned above, plants harbouring reduced ADH1 expression exhibitincrease tolerance to allyl alcohol. Therefore, bombarded plants formodified miRNA to target ADH1 were regenerated in media containing 30 mMallyl alcohol and compared to the regeneration rate of control plants.118 GEiGS #3+SWAP11 allyl alcohol selected plants survived, compared to51 control plants on allyl alcohol media (data not shown). Of theselected GEiGS #3+SWAP11, 5 were shown to harbour the DONOR (data notshown). The large amount of plants regenerating in the DONOR-treatedplants, might be due to transient expression, during the bombardmentprocess, as well.

Thus, PDS3 and ADH1 selection through bleached phenotype (FIG. 16) andallyl alcohol selection (FIG. 17), respectively, give an ideal means fortransformed plantlet selection for genotyping.

Swap region of 4 kb was assessed primarily through internal primers andspecific amplicon differentiation of original wild type to insertion viarestriction enzyme digestion variation.

ADH1 (FIG. 14) showed a comparative genotype of allyl alcohol selectedplants with the expected DONOR presence restriction pattern whencompared to restricted and non-restricted DONOR plasmid. PDS3 (FIG. 13)showed a comparison of bombarded samples phenotypes with and withoutDONOR and their respective differential restriction enzyme digestionpatterns compared to that of restricted and non-restricted DONORplasmid. These results provided a clear association of PDS3albino/bleached phenotype to the expected restriction pattern.Subsequent external PCR combining specific internal, within the Swapregion, in conjunction with external primer, outside and specific to thegenomic region to swap into was carried out (data not shown). Furthervalidation of the Swap was obtained through Sanger sequencing of the PCRamplicons, in order to assess heterozygous, homozygous, or presence ofDONOR Swap (data not shown).

Example 12 Modified miRNA Reduce the Expression of their New Target Gene

In order to verify the potential of the modified miRNAs in the GEiGSsystem to down regulate the expression of their newly designatedtargets, gene expression analysis was carried out using qRT-PCR(quantitative Real-Time PCR). RNA was extracted and reverse transcribed,from the positively identified regenerated plants and compared toregenerated plants, treated in parallel, but were not introduced withthe relevant modifying constructs. In the case, where miR-173 wasmodified to target PDS3 (GEiGS #4+SWAP4), a reduction of 83% in the geneexpression level, on average, was observed (FIG. 15). In plants withmodified miR-390 to target ADH1 (GEiGS #3+SWAP11), a similar change ingene expression was observed, 82% of the levels in the control plants(FIG. 16). Taken together, these results substantiate the gene editingmethods of modifying endogenous miRNAs to successfully target new genesand reduce their expression, by replacing the target recognitionsequence in the miRNA transcript in the endogenous locus.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by into thespecification, to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting.

1. (canceled)
 2. A method of modifying a gene encoding or processed intoan RNA silencing molecule to a target RNA in a eukaryotic cell, with theproviso that said eukaryotic cell is not a plant cell, the methodcomprising introducing into the eukaryotic cell a DNA editing agentwhich redirects a silencing specificity of said RNA silencing moleculetowards a second target RNA, said target RNA and said second target RNAbeing distinct, thereby modifying the gene encoding the RNA silencingmolecule.
 3. (canceled)
 4. The method of claim 2, wherein the geneencoding the RNA silencing molecule is endogenous to the eukaryoticcell.
 5. (canceled)
 6. The method of claim 2, wherein said modifyingsaid gene encoding said RNA silencing molecule comprises imparting saidRNA silencing molecule with at least 45% complementarity towards saidsecond target RNA. 7-13. (canceled)
 14. The method of claim 2, whereinsaid said RNA silencing molecule is processed from a precursor.
 15. Themethod of claim 2, wherein said RNA silencing molecule is a RNAinterference (RNAi) molecule.
 16. The method of claim 15, wherein saidRNAi molecule is selected from the group consisting of a smallinterfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA(miRNA), a Piwi-interacting RNA (piRNA) and trans-acting siRNA(tasiRNA).
 17. (canceled)
 18. The method of claim 15, wherein said RNAimolecule is modified to preserve secondary RNA structure and to berecognized by cellular RNAi factors.
 19. The method of claim 2, whereinsaid modifying said gene is affected by a modification selected from thegroup consisting of a deletion, an insertion, a point mutation and acombination thereof. 20-24. (canceled)
 25. The method of claim 19,wherein said modification comprises a modification of at most 200nucleotides.
 26. The method of claim 19, wherein said method furthercomprises introducing into said eukaryotic cell a donor oligonucleotidesequence.
 27. The method of claim 2, wherein said DNA editing agentcomprises at least one gRNA.
 28. (canceled)
 29. The method of claim 2,wherein said DNA editing agent comprises an endonuclease.
 30. The methodof claim 29, wherein said DNA editing agent comprises a DNA editingsystem selected from the group consisting of a meganuclease, a zincfinger nucleases (ZFN), a transcription-activator like effector nuclease(TALEN) and CRISPR.
 31. The method of claim 29, wherein saidendonuclease comprises Cas9. 32-34. (canceled)
 35. The method of claim2, wherein said second target RNA is endogenous or exogenous to saideukaryotic cell.
 36. The method of claim 35, wherein said second targetRNA is associated with a cancer or with an infectious disease. 37-42.(canceled)
 43. A method of treating an infectious disease, a monogenicrecessive disorder, an autoimmune disease or a cancerous disease in asubject in need thereof, the method comprising modifying a gene encodingor processed into an RNA silencing molecule according to the method ofclaim 2, wherein said second target RNA is associated with onset orprogression of said infectious disease, said monogenic recessivedisorder, said autoimmune disease or said cancerous disease, therebytreating the infectious disease in the subject. 44-46. (canceled)
 47. Amethod of enhancing efficacy and/or specificity of a chemotherapeuticagent in a subject in need thereof, the method comprising modifying agene encoding or processed into an RNA silencing molecule according tothe method of claim 2, wherein said second target RNA is associated withenhancement of efficacy and/or specificity of said chemotherapeuticagent, thereby enhancing efficacy and/or specificity of achemotherapeutic agent in the subject.
 48. A method of inducing cellapoptosis in a subject in need thereof, the method comprising modifyinga gene encoding or processed into an RNA silencing molecule according tothe method of claim 2, wherein said second target RNA is associated withsaid apoptosis, thereby inducing cell apoptosis in the subject.
 49. Amethod of generating a eukaryotic non-human organism, with the provisothat said organism is not a plant, wherein at least some of the cells ofsaid organism comprise a modified gene encoding or processed into a RNAsilencing molecule comprising a silencing specificity towards a secondtarget RNA, the method comprising modifying a gene according to themethod of claim 2, thereby generating the eukaryotic non-human organism.50. A method of modifying a gene encoding or processed into a non-codingRNA molecule having no RNA silencing activity in a eukaryotic cell, withthe proviso that said eukaryotic cell is not a plant cell, the methodcomprising introducing into the eukaryotic cell a DNA editing agentconferring a silencing specificity of said non-coding RNA moleculetowards a target RNA of interest, thereby modifying the gene encoding orprocessed into the non-coding RNA molecule.